53. A method comprising:(a) testing the compound of claim 31 for
inhibition of gene expression of a gene encoding an RNA comprising a
lysine riboswitch, wherein the inhibition is via the lysine
riboswitch,(b) inhibiting gene expression by bringing into contact a cell
and a compound that inhibited gene expression in step (a),wherein the
cell comprises a gene encoding an RNA comprising the lysine
riboswitch,wherein the compound inhibits expression of the gene by
binding to the lysine riboswitch.

54. The method of claim 1, wherein the compound of formula I is selected
from Compounds 1, 2, 3, 4 and 7 of FIG. 2a-1 and 2a-II.3.

55. The method of claim 1, wherein the compound of Formula I is Compounds
1, 2 or 4.

56. The compound of claim 31, wherein the compound of formula I is
selected from Compounds 1, 2, 3, 4 and 7 of FIG. 2a-1 and 2a-II.3

57. The compound of claim 31, wherein the compound of Formula I is
Compounds 1, 2 or 4.

[0003]The disclosed invention is generally in the field of gene expression
and specifically in the area of regulation of gene expression.

BACKGROUND OF THE INVENTION

[0004]Precision genetic control is an essential feature of living systems,
as cells must respond to a multitude of biochemical signals and
environmental cues by varying genetic expression patterns. Most known
mechanisms of genetic control involve the use of protein factors that
sense chemical or physical stimuli and then modulate gene expression by
selectively interacting with the relevant DNA or messenger RNA sequence.
Proteins can adopt complex shapes and carry out a variety of functions
that permit living systems to sense accurately their chemical and
physical environments. Protein factors that respond to metabolites
typically act by binding DNA to modulate transcription initiation (e.g.
the lac repressor protein; Matthews, K. S., and Nichols, J. C., 1998,
Prog. Nucleic Acids Res. Mol. Biol. 58, 127-164) or by binding RNA to
control either transcription termination (e.g. the PyrR protein; Switzer,
R. L., et al., 1999, Prog. Nucleic Acids Res. Mol. Biol. 62, 329-367) or
translation (e.g. the TRAP protein; Babitzke, P., and Gollnick, P., 2001,
J. Bacteriol. 183, 5795-5802). Protein factors respond to environmental
stimuli by various mechanisms such as allosteric modulation or
post-translational modification, and are adept at exploiting these
mechanisms to serve as highly responsive genetic switches (e.g. see
Ptashne, M., and Gann, A. (2002). Genes and Signals. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).

[0005]In addition to the widespread participation of protein factors in
genetic control, it is also known that RNA can take an active role in
genetic regulation. Recent studies have begun to reveal the substantial
role that small non-coding RNAs play in selectively targeting mRNAs for
destruction, which results in down-regulation of gene expression (e.g.
see Hannon, G. J. 2002, Nature 418, 244-251 and references therein). This
process of RNA interference takes advantage of the ability of short RNAs
to recognize the intended mRNA target selectively via Watson-Crick base
complementation, after which the bound mRNAs are destroyed by the action
of proteins. RNAs are ideal agents for molecular recognition in this
system because it is far easier to generate new target-specific RNA
factors through evolutionary processes than it would be to generate
protein factors with novel but highly specific RNA binding sites.

[0007]Bacterial riboswitch RNAs are genetic control elements that are
located primarily within the 5'-untranslated region (5''-UTR) of the main
coding region of a particular mRNA. Structural probing studies (discussed
further below) reveal that riboswitch elements are generally composed of
two domains: a natural aptamer (T. Hermann, D. J. Patel, Science 2000,
287, 820; L. Gold, et al., Annual Review of Biochemistry 1995, 64, 763)
that serves as the ligand-binding domain, and an `expression platform`
that interfaces with RNA elements that are involved in gene expression
(e.g. Shine-Dalgarno (SD) elements; transcription terminator stems). What
is needed in the art are methods and compositions that can be used to
regulate lysine riboswitches.

BRIEF SUMMARY OF THE INVENTION

[0008]It has been discovered that certain natural mRNAs serve as
metabolite-sensitive genetic switches wherein the RNA directly binds a
small organic molecule. This binding process changes the conformation of
the mRNA, which causes a change in gene expression by a variety of
different mechanisms. The natural switches are targets for antibiotics
and other small molecule therapies.

[0009]Disclosed are compounds, and compositions containing such compounds,
that can activate, deactivate or block the lysine riboswitch. Also
disclosed are compositions and methods for activating, deactivating or
blocking the lysine riboswitch. Riboswitches function to control gene
expression through the binding or removal of a trigger molecule.
Compounds can be used to activate, deactivate or block a riboswitch. The
trigger molecule for a riboswitch (as well as other activating compounds)
can be used to activate a riboswitch. Compounds other than the trigger
molecule generally can be used to deactivate or block a riboswitch.
Riboswitches can also be deactivated by, for example, removing trigger
molecules from the presence of the riboswitch. A riboswitch can be
blocked by, for example, binding of an analog of the trigger molecule
that does not activate the riboswitch.

[0010]Also disclosed are compositions and methods for altering expression
of an RNA molecule, or of a gene encoding an RNA molecule, where the RNA
molecule includes a lysine riboswitch, by bringing a compound into
contact with the RNA molecule. Riboswitches function to control gene
expression through the binding or removal of a trigger molecule. Thus,
subjecting an RNA molecule of interest that includes a lysine riboswitch
to conditions that activate, deactivate or block the riboswitch can be
used to alter expression of the RNA. Expression can be altered as a
result of, for example, termination of transcription or blocking of
ribosome binding to the RNA. Binding of a trigger molecule or an analog
thereof can, depending on the nature of the riboswitch, reduce or prevent
expression of the RNA molecule or promote or increase expression of the
RNA molecule.

[0011]Also disclosed are compositions and methods for regulating
expression of a naturally occurring gene or RNA that contains a lysine
riboswitch by activating, deactivating or blocking the riboswitch. If the
gene is essential for survival of a cell or organism that harbors it,
activating, deactivating or blocking the lysine riboswitch can result in
death, stasis or debilitation of the cell or organism. For example,
activating a naturally occurring riboswitch in a naturally occurring gene
that is essential to survival of a microorganism can result in death of
the microorganism (if activation of the riboswitch turns off or represses
expression). This is one basis for the use of the disclosed compounds and
methods for antimicrobial and antibiotic effects.

[0012]Disclosed herein is a method of inhibiting gene expression, the
method comprising (a) bringing into contact a compound and a cell, (b)
wherein the compound has the structure of Formula I:

##STR00001##

[0013]wherein R2 and R3 are each independently positively
charged, can serve as a hydrogen bond donor, or both,

[0014]wherein R1 is negatively charged, R4 is negatively
charged, or R1 and R4 are in a resonance hybrid with a net
negative charge,

[0020]wherein can each independently represent a single or double bond,
and

[0021]wherein the compound is not lysine, and wherein the cell comprises a
gene encoding an RNA comprising a lysine-responsive riboswitch, wherein
the compound inhibits expression of the gene by binding to the
lysine-responsive riboswitch.

[0022]R3 can be positively charged and can serve as a hydrogen bond
donor. R5 can be uncharged. R9 can be C, O, or S. The pKa
of R3 can be 7 or higher. R13 can be positively charged, and
can serve as a hydrogen bond donor, or both.

[0023]In one example, R6, R7, R8, R9, R10 and
R11 are not all simultaneously C, CH, or CH2.

[0024]In another example, R1, R2, R3, R4 and R9
are not simultaneously O, NH3.sup.+, NH3.sup.+, O and S,
respectively. Furthermore, in another example, R1, R2, R3,
and R4 are not simultaneously O, H, NH3.sup.+, and O,
respectively. In another example, R1, R2, R3, R4 and
R9 are not simultaneously CO2-, NH3.sup.+,
NH3.sup.+, and H, respectively. In a further example, R1,
R2, R3, R4 and R11 are not simultaneously O,
NH3.sup.+, NH3.sup.+, O and C--CO2-, respectively. In
a further example, R1, R2, R3, and R4 are not
simultaneously NHOH, NH3.sup.+, NH3.sup.+, O and S,
respectively.

[0028]In a further example, R10 can be N, NH, O, or S. In a further
example, R7 can be CH.

[0029]The cell can be identified as being in need of inhibited gene
expression. The cell can be a bacterial cell, for example, and the
compound can kill or inhibit the growth of the bacterial cell. The
compound and the cell can be brought into contact by administering the
compound to a subject. In one example, the compound is not a substrate
for enzymes of the subject that have lysine as a substrate. The compound
can also not be a substrate for enzymes of the subject that alter lysine.
The compound can also not be a substrate for enzymes of the subject that
metabolize lysine. The compound can also not be a substrate for enzymes
of the subject that catabolize lysine. The cell can be a bacterial cell
in the subject, wherein the compound kills or inhibits the growth of the
bacterial cell.

[0030]Disclosed herein is a compound having the structure of Formula I:

##STR00002##

[0031]wherein R2 and R3 are each independently positively
charged, can serve as a hydrogen bond donor, or both,

[0032]wherein R1 is negatively charged, R4 is negatively
charged, or R1 and R4 are in a resonance hybrid with a net
negative charge,

[0039]wherein can each independently represent a single or double bond,
and

[0040]wherein the compound is not lysine.

[0041]R3 can be positively charged and can serve as a hydrogen bond
donor. R5 can be uncharged. R9 can be C, O, or S. The pKa
of R3 can be 7 or higher. R13 can be positively charged, and
can serve as a hydrogen bond donor, or both.

[0042]In one example, R6, R7, R8, R9, R10 and
R11 are not all simultaneously C, CH, or CH2.

[0043]In another example, R1, R2, R3, R4 and R9
are not simultaneously O, NH3.sup.+, NH3.sup.+, O and S,
respectively. Furthermore, in another example, R1, R2, R3,
and R4 are not simultaneously O, H, NH3.sup.+, and O,
respectively. In another example, R1, R2, R3, R4 and
R9 are not simultaneously CO2-, NH3.sup.+,
NH3.sup.+, and H, respectively. In a further example, R1,
R2, R3, R4 and R11 are not simultaneously O,
NH3.sup.+, NH3.sup.+, O and C--CO2-, respectively. In
a further example, R1, R2, R3, and R4 are not
simultaneously NHOH, NH3.sup.+, NH3.sup.+, O and S,
respectively.

[0047]In a further example, R10 can be N, NH, O, or S. In a further
example, R7 can be CH.

[0048]Further disclosed is a composition comprising the compound described
above and a regulatable gene expression construct comprising a nucleic
acid molecule encoding an RNA comprising a lysine riboswitch operably
linked to a coding region, wherein the lysine riboswitch regulates
expression of the RNA, wherein the lysine riboswitch and coding region
are heterologous. The lysine riboswitch can produce a signal when
activated by the compound. For example, the riboswitch can change
conformation when activated by the compound, and the change in
conformation can produce a signal via a conformation dependent label.
Furthermore, the riboswitch can change conformation when activated by the
compound, wherein the change in conformation causes a change in
expression of the coding region linked to the riboswitch, wherein the
change in expression produces a signal. The signal can be produced by a
reporter protein expressed from the coding region linked to the
riboswitch.

[0049]Also disclosed is a method comprising: (a) testing the compound as
described above for inhibition of gene expression of a gene encoding an
RNA comprising a lysine riboswitch, wherein the inhibition is via the
lysine riboswitch, and (b) inhibiting gene expression by bringing into
contact a cell and a compound that inhibited gene expression in step (a),
wherein the cell comprises a gene encoding an RNA comprising the lysine
riboswitch, wherein the compound inhibits expression of the gene by
binding to the lysine riboswitch.

[0050]Further disclosed is a method of inhibiting the growth of and/or
killing bacteria, comprising contacting the bacteria with a compound
disclosed above. Disclosed herein is also a method of inhibiting growth
of a cell, such as a bacterial cell, that is in a subject, the method
comprising administering an effective amount of a compound as disclosed
herein to the subject. This can result in the compound being brought into
contact with the cell. The subject can have, for example, a bacterial
infection, and the bacterial cells can be the cells to be inhibited by
the compound. The bacteria can be any bacteria. Bacterial growth can also
be inhibited in any context in which bacteria are found. For example,
bacterial growth in fluids, biofilms, and on surfaces can be inhibited.
The compounds disclosed herein can be administered or used in combination
with any other compound or composition. For example, the disclosed
compounds can be administered or used in combination with another
antimicrobial compound.

[0051]Also disclosed are compositions and methods for selecting and
identifying compounds that can activate, deactivate or block a
riboswitch. Activation of a riboswitch refers to the change in state of
the riboswitch upon binding of a trigger molecule. A riboswitch can be
activated by compounds other than the trigger molecule and in ways other
than binding of a trigger molecule. The term trigger molecule is used
herein to refer to molecules and compounds that can activate a
riboswitch. This includes the natural or normal trigger molecule for the
riboswitch and other compounds that can activate the riboswitch. Natural
or normal trigger molecules are the trigger molecule for a given
riboswitch in nature or, in the case of some non-natural riboswitches,
the trigger molecule for which the riboswitch was designed or with which
the riboswitch was selected (as in, for example, in vitro selection or in
vitro evolution techniques). Non-natural trigger molecules can be
referred to as non-natural trigger molecules.

[0052]Deactivation of a riboswitch refers to the change in state of the
riboswitch when the trigger molecule is not bound. A riboswitch can be
deactivated by binding of compounds other than the trigger molecule and
in ways other than removal of the trigger molecule. Blocking of a
riboswitch refers to a condition or state of the riboswitch where the
presence of the trigger molecule does not activate the riboswitch.
Activation of a riboswitch can be assessed in any suitable manner. For
example, the riboswitch can be linked to a reporter RNA and expression,
expression level, or change in expression level of the reporter RNA can
be measured in the presence and absence of the test compound. As another
example, the riboswitch can include a conformation dependent label, the
signal from which changes depending on the activation state of the
riboswitch. Such a riboswitch preferably uses an aptamer domain from or
derived from a naturally occurring riboswitch. As can be seen, assessment
of activation of a riboswitch can be performed with the use of a control
assay or measurement or without the use of a control assay or
measurement. Methods for identifying compounds that deactivate a
riboswitch can be performed in analogous ways.

[0053]Also disclosed are compounds made by identifying a compound that
activates, deactivates or blocks a riboswitch and manufacturing the
identified compound. This can be accomplished by, for example, combining
compound identification methods as disclosed elsewhere herein with
methods for manufacturing the identified compounds. For example,
compounds can be made by bringing into contact a test compound and a
riboswitch, assessing activation of the riboswitch, and, if the
riboswitch is activated by the test compound, manufacturing the test
compound that activates the riboswitch as the compound.

[0054]Also disclosed are compounds made by checking activation,
deactivation or blocking of a riboswitch by a compound and manufacturing
the checked compound. This can be accomplished by, for example, combining
compound activation, deactivation or blocking assessment methods as
disclosed elsewhere herein with methods for manufacturing the checked
compounds. For example, compounds can be made by bringing into contact a
test compound and a riboswitch, assessing activation of the riboswitch,
and, if the riboswitch is activated by the test compound, manufacturing
the test compound that activates the riboswitch as the compound. Checking
compounds for their ability to activate, deactivate or block a riboswitch
refers to both identification of compounds previously unknown to
activate, deactivate or block a riboswitch and to assessing the ability
of a compound to activate, deactivate or block a riboswitch where the
compound was already known to activate, deactivate or block the
riboswitch.

[0055]Disclosed herein is also a method of inhibiting growth of a cell,
such as a bacterial cell, that is in a subject, the method comprising
administering an effective amount of a compound as disclosed herein to
the subject. This can result in the compound being brought into contact
with the cell. The subject can have, for example, a bacterial infection,
and the bacterial cells can be the cells to be inhibited by the compound.
The bacteria can be any bacteria, such as bacteria from the genus
Bacillus, Actinobacillus, Clostridium, Desulfitobacterium, Enterococcus,
Erwinia, Escherichia, Exiguobacterium, Fusobacterium, Geobacillus,
Haemophilus, Idiomarina, Lactobacillus, Lactococcus, Leuconostoc,
Listeria, Moorella, Oceanobacillus, Oenococcus, Pasteurella, Pediococcus,
Shewanella, Shigella, Solibacter, Staphylococcus, Thermoanaerobacter,
Thermotoga, and Vibrio, for example. Bacterial growth can also be
inhibited in any context in which bacteria are found. For example,
bacterial growth in fluids, biofilms, and on surfaces can be inhibited.
The compounds disclosed herein can be administered or used in combination
with any other compound or composition. For example, the disclosed
compounds can be administered or used in combination with another
antimicrobial compound.

[0056]Additional advantages of the disclosed method and compositions will
be set forth in part in the description which follows, and in part will
be understood from the description, or can be learned by practice of the
disclosed method and compositions. The advantages of the disclosed method
and compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims. It is
to be understood that both the foregoing general description and the
following detailed description are exemplary and explanatory only and are
not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057]The accompanying drawings, which are incorporated in and constitute
a part of this specification, illustrate several embodiments of the
disclosed method and compositions and together with the description,
serve to explain the principles of the disclosed method and compositions.

[0058]FIG. 1 shows the structure and function of the lysC riboswitch from
B. subtilis. (a) The sequence and secondary structure model of the
repressed-state lysC 5'-UTR from B. subtilis. Certain nucleotides are
conserved in at least 90% of the representatives identified by
bioinformatics. The putative antiterminator hairpin that forms in the
absence of ligand is also shown. An additional 63 nucleotides reside
between nucleotide 268 and the lysC start codon. A 179-nucleotide
construct (179 lysC) spanning nucleotides 27 through 205 (bracketed) was
used to determine ligand binding affinities. Nucleotides where
spontaneous cleavage activity changes upon ligand binding are encircled
(regions A, B, and C) and correspond to the bands identified in FIG. 2b.
(b) The lysine biosynthesis pathway in B. subtilis. The name of the gene
that codes for the enzyme or transporter at each step is indicated
adjacent to a solid arrow. The expression of aspartokinase II and a
lysine-specific importer (lysC and yvsH, boxed) is regulated by a lysine
riboswitch in the 5'-UTR of each gene. IUPAC numbering for each carbon
atom of lysine is shown. The lysine analog L-aminoethylcysteine (AEC,
boxed), which differs from lysine in that C4 is replaced by sulfur, is
also depicted.

[0059]FIG. 2 shows molecular recognition by a lysine riboswitch receptor.
(a) Chemical structures of lysine analogs examined in this study for
binding to 179 lysC. The expected protonation states under the conditions
used for in-line probing are shown for each functional group. Shaded
regions highlight the functional groups that differ from lysine. KD
values for the interaction of each analog with 179 lysC are the average
of two independent repeats yielding the standard deviation shown. (b)
Representative denaturing polyacrylamide gel separating products
generated by in-line probing analysis of 179 lysC with 2. Concentrations
of L-4-oxalysine (2) used ranged from 1 nM to 6 mM. (NR) denotes
untreated, full-length RNA, and (-) represents the reaction in the
absence of any added compound. The length of each product band was
determined by comparison to a partial digest with RNase T1 (T1) and a
partial digest with alkalai (-OH). Numbered bands correspond to
selected products of RNAse T1 digestion (G-specific cleavage). (c) Plot
depicting the normalized fraction of RNA cleaved at regions A, B, and C
versus the concentration of either lysine or L-4-oxalysine (2). The
curves indicate the best fit of the data to an equation for a two-state
binding model (Example 1). (d) Schematic representation of the molecular
recognition characteristics of the lysine riboswitch.

[0060]FIG. 3 shows lysine derivatives that inhibit bacterial growth and
repress gene expression. (a) The growth of B. subtilis in a
chemically-defined minimal media (Example 1) was monitored in the
presence of 100 μM of each lysine derivative (compounds are numbered
as in FIG. 2) or in the absence of any added compound (-) by measuring
absorbance of the culture at 595 nm. Growth was not significantly
inhibited by the compounds that are not shown. (b) Plot of bacterial
growth in the presence of 100 μM of each lysine analog after 6 h,
normalized to the growth in the absence of added compound (-). Circles
highlight lysine analogs that bind the riboswitch with a KD below 13
μM. (c) The minimum concentration of each compound required to
completely inhibit growth (MIC) over 24 h. Also shown is the expression
of a β-galactosidase gene fused to a second copy of the lysC
riboswitch in a wild-type B. subtilis strain after growing for 3 h in the
presence of 5 mM of each indicated derivative (Miller units). The
compounds highlighted in grey bind the riboswitch, fully inhibit growth,
and repress gene expression. (d) β-galactosidase expression as
described in c as a function of increasing concentrations of lysine, 1,
or 2 from 0.3 μM to 1 mM. Relative expression denotes the Miller units
at each concentration relative to the Miller units in the absence of any
added compound. The arrows indicate the MIC of 1 and 2.

[0061]FIG. 4 shows mutations within the lysine riboswitch confer
resistance to lysine derivatives and deregulate the lysine riboswitch.
(a) Nucleotide changes in the M1 and M2 mutants identified in the B.
subtilis lysC riboswitch are boxed. Changes in the in-line probing
pattern caused by each mutation (shown in c) are encircled. (b) The MIC
values are given for each compound toward a wild-type, M1, or M2 strain
of B. subtilis. KD values are for the interaction of the indicated
compound with the 179-nucleotide receptor domain of the wild-type, M1 or
M2 riboswitches at 37° C., and Miller units indicate the
expression of a β-galactosidase gene controlled by a lysine
riboswitch with no mutation or with the M1 or M2 mutation. In each case,
the reporter gene was expressed in a wild-type B. subtilis strain while
growing for 3 h in the presence of the indicated lysine derivative at 5
mM (WT) or 1 mM (M1 and M2). (c) In-line probing analysis of the
179-nucleotide lysC receptor domain with either the M1 or M2 mutation at
the indicated temperatures. The changes in the spontaneous cleavage
pattern induced by each mutation are highlighted (additional details are
as described in the legend to FIG. 2b). (d) In vitro transcription
analysis of the DNA templates corresponding to the wild-type, M1, or M2
lysine riboswitches. Reactions were conducted with E. coli RNA polymerase
holoenzyme in the presence (+) or absence (-) of 10 mM lysine as
indicated for each lane. Below each lane is noted the percentage of
transcription termination (T) at the expected site (FIG. 1a) relative to
the total amount of terminated plus full length (FL) RNA. (e) In vitro
transcription analysis of the lysine riboswitch as a function of an
increasing concentration of the indicated compound. The apparent
concentration at which termination efficiency is half-maximally attained
(T50) by lysine is depicted with a dashed line for the WT or M2
riboswitch.

[0062]FIG. 5 shows the consensus sequence and secondary structure for the
lysine riboswitch, determined by comparing the sequences of all known
examples of the lysine riboswitch.

[0063]FIG. 6 shows the pathways for lysine biosynthesis and import in
bacteria. Escherichia coli gene names are used throughout, with the
exception of the underlined gene names, which are found in Bacillus
subtilis, and the names of the putative lysine transporter genes (boxed).
Most bacterial species convert tetrahydrodipicolinate to
L,L-diaminopimelate via two N-succinyl intermediates, catalyzed by the
products of the dapD, dapC, and dapE genes in E. coli. Some species,
including B. subtilis, accomplish this conversion via N-acetyl
intermediates, catalyzed by the products of the dapD, patA, and ykuR
genes. In addition to synthesizing lysine, many bacteria also import
lysine from their environment. The well-characterized lysine-specific
importer, coded by lysP in E. coli, several Gram negative and Gram
positive species, In addition, three other putative lysine transporters
were recently identified by comparative analysis of genes regulated by
lysine riboswitches. The yvsH gene of B. subtilis codes for a putative
lysine transporter with high sequence similarity to the APA basic amino
acid/polyamine antiporter family. The lys W class of genes, found in
Vibrio and Shewanella species, code for a putative transporter with high
sequence similarity to the NhaC Na+:H+ antiporter superfamily. The lysXY
class of putative lysine transporters has high sequence similarity to an
ATP-dependent transport system for other amino acids.

[0064]FIG. 7 shows growth of B. subtilis lysine auxotroph strain 1A40 upon
supplementation of minimal media with various compounds as indicated.
Both compound 3 (see FIG. 2 for compound identities) and lysine support
growth in a chemically-defined minimal media (Example 1). Growth was
established by measuring the absorbance at 600 nm after 3 h in the
presence of 1 mM of the compounds indicated or in the absence of added
compound (-).

DETAILED DESCRIPTION OF THE INVENTION

[0065]The disclosed methods and compositions can be understood more
readily by reference to the following detailed description of particular
embodiments and the Examples included therein and to the Figures and
their previous and following description.

[0066]Messenger RNAs are typically thought of as passive carriers of
genetic information that are acted upon by protein- or small
RNA-regulatory factors and by ribosomes during the process of
translation. It was discovered that certain mRNAs carry natural aptamer
domains and that binding of specific metabolites directly to these RNA
domains leads to modulation of gene expression. Natural riboswitches
exhibit two surprising functions that are not typically associated with
natural RNAs. First, the mRNA element can adopt distinct structural
states wherein one structure serves as a precise binding pocket for its
target metabolite. Second, the metabolite-induced allosteric
interconversion between structural states causes a change in the level of
gene expression by one of several distinct mechanisms. Riboswitches
typically can be dissected into two separate domains: one that
selectively binds the target (aptamer domain) and another that influences
genetic control (expression platform). It is the dynamic interplay
between these two domains that results in metabolite-dependent allosteric
control of gene expression.

[0067]Distinct classes of riboswitches have been identified and are shown
to selectively recognize activating compounds (referred to herein as
trigger molecules). For example, coenzyme B12, glycine, thiamine
pyrophosphate (TPP), and flavin mononucleotide (FMN) activate
riboswitches present in genes encoding key enzymes in metabolic or
transport pathways of these compounds. The aptamer domain of each
riboswitch class conforms to a highly conserved consensus sequence and
structure. Thus, sequence homology searches can be used to identify
related riboswitch domains. Riboswitch domains have been discovered in
various organisms from bacteria, archaea, and eukarya.

[0068]Lysine riboswitches are bacterial RNA structures that sense the
concentration of lysine and regulate the expression of lysine
biosynthesis and transport genes. Members of this riboswitch class are
found in the 5'-untranslated region (5'-UTR) of messenger RNAs, where
they form highly selective receptors for lysine. Lysine binding to the
receptor stabilizes an mRNA tertiary structure that, in most cases,
causes transcription termination before the adjacent open reading frame
can be expressed. A lysine riboswitch can be used for antibacterial
therapy by designing compounds that bind the riboswitch and suppress
lysine biosynthesis and transport genes. Lysine analogs that bind to
riboswitches and thereby inhibit bacterial growth have been identified,
and their mechanism of action elucidated (Example 1).

A. General Organization of Riboswitch RNAs

[0069]Bacterial riboswitch RNAs are genetic control elements that are
located primarily within the 5'-untranslated region (5'-UTR) of the main
coding region of a particular mRNA. Structural probing studies (discussed
further below) reveal that riboswitch elements are generally composed of
two domains: a natural aptamer (T. Hermann, D. J. Patel, Science 2000,
287, 820; L. Gold, et al., Annual Review of Biochemistry 1995, 64, 763)
that serves as the ligand-binding domain, and an `expression platform`
that interfaces with RNA elements that are involved in gene expression
(e.g. Shine-Dalgarno (SD) elements; transcription terminator stems).
These conclusions are drawn from the observation that aptamer domains
synthesized in vitro bind the appropriate ligand in the absence of the
expression platform (see Examples 2, 3 and 6 of U.S. Application
Publication No. 2005-0053951). Moreover, structural probing
investigations suggest that the aptamer domain of most riboswitches
adopts a particular secondary- and tertiary-structure fold when examined
independently, that is essentially identical to the aptamer structure
when examined in the context of the entire 5' leader RNA. This indicates
that, in many cases, the aptamer domain is a modular unit that folds
independently of the expression platform (see Examples 2, 3 and 6 of U.S.
Application Publication No. 2005-0053951).

[0070]Ultimately, the ligand-bound or unbound status of the aptamer domain
is interpreted through the expression platform, which is responsible for
exerting an influence upon gene expression. The view of a riboswitch as a
modular element is further supported by the fact that aptamer domains are
highly conserved amongst various organisms (and even between kingdoms as
is observed for the TPP riboswitch), (N. Sudarsan, et al., RNA 2003, 9,
644) whereas the expression platform varies in sequence, structure, and
in the mechanism by which expression of the appended open reading frame
is controlled. For example, ligand binding to the TPP riboswitch of the
tenA mRNA of B. subtilis causes transcription termination (A. S. Mironov,
et al., Cell 2002, 111, 747). This expression platform is distinct in
sequence and structure compared to the expression platform of the TPP
riboswitch in the thiM mRNA from E. coli, wherein TPP binding causes
inhibition of translation by a SD blocking mechanism (see Example 2 of
U.S. Application Publication No. 2005-0053951). The TPP aptamer domain is
easily recognizable and of near identical functional character between
these two transcriptional units, but the genetic control mechanisms and
the expression platforms that carry them out are very different.

[0071]Aptamer domains for riboswitch RNAs typically range from ˜70
to 170 nt in length (FIG. 11 of U.S. Application Publication No.
2005-0053951). This observation was somewhat unexpected given that in
vitro evolution experiments identified a wide variety of small
molecule-binding aptamers, which are considerably shorter in length and
structural intricacy (T. Hermann, D. J. Patel, Science 2000, 287, 820; L.
Gold, et al., Annual Review of Biochemistry 1995, 64, 763; M. Famulok,
Current Opinion in Structural Biology 1999, 9, 324). Although the reasons
for the substantial increase in complexity and information content of the
natural aptamer sequences relative to artificial aptamers remains to be
proven, this complexity is believed required to form RNA receptors that
function with high affinity and selectivity. Apparent KD values for
the ligand-riboswitch complexes range from low nanomolar to low
micromolar. It is also worth noting that some aptamer domains, when
isolated from the appended expression platform, exhibit improved affinity
for the target ligand over that of the intact riboswitch. (˜10 to
100-fold) (see Example 2 of U.S. Application Publication No.
2005-0053951). Presumably, there is an energetic cost in sampling the
multiple distinct RNA conformations required by a fully intact riboswitch
RNA, which is reflected by a loss in ligand affinity. Since the aptamer
domain must serve as a molecular switch, this might also add to the
functional demands on natural aptamers that might help rationalize their
more sophisticated structures.

B. Riboswitch Regulation of Transcription Termination in Bacteria

[0072]Bacteria primarily make use of two methods for termination of
transcription. Certain genes incorporate a termination signal that is
dependent upon the Rho protein, (J. P. Richardson, Biochimica et
Biophysica Acta 2002, 1577, 251). while others make use of
Rho-independent terminators (intrinsic terminators) to destabilize the
transcription elongation complex (I. Gusarov, E. Nudler, Molecular Cell
1999, 3, 495; E. Nudler, M. E. Gottesman, Genes to Cells 2002, 7, 755).
The latter RNA elements are composed of a GC-rich stem-loop followed by a
stretch of 6-9 uridyl residues. Intrinsic terminators are widespread
throughout bacterial genomes (F. Lillo, et al., 2002, 18, 971), and are
typically located at the 3'-termini of genes or operons. Interestingly,
an increasing number of examples are being observed for intrinsic
terminators located within 5'-UTRs.

[0073]Amongst the wide variety of genetic regulatory strategies employed
by bacteria there is a growing class of examples wherein RNA polymerase
responds to a termination signal within the 5'-UTR in a regulated fashion
(T. M. Henkin, Current Opinion in Microbiology 2000, 3, 149). During
certain conditions the RNA polymerase complex is directed by external
signals either to perceive or to ignore the termination signal. Although
transcription initiation might occur without regulation, control over
mRNA synthesis (and of gene expression) is ultimately dictated by
regulation of the intrinsic terminator. Presumably, one of at least two
mutually exclusive mRNA conformations results in the formation or
disruption of the RNA structure that signals transcription termination. A
trans-acting factor, which in some instances is a RNA (F. J. Grundy, et
al., Proceedings of the National Academy of Sciences of the United States
of America 2002, 99, 11121; T. M. Henkin, C. Yanofsky, Bioessays 2002,
24, 700) and in others is a protein (J. Stulke, Archives of Microbiology
2002, 177, 433), is generally required for receiving a particular
intracellular signal and subsequently stabilizing one of the RNA
conformations. Riboswitches offer a direct link between RNA structure
modulation and the metabolite signals that are interpreted by the genetic
control machinery.

[0074]Most clinical antibacterial compounds target one of only four
cellular processes (Wolfson 2006). Since bacteria have well developed
resistance mechanisms to protect these processes (D'Costa 2006), it is
useful to discover new targets that are vulnerable to drug intervention.
One type of vulnerable process is the regulation of gene expression by
riboswitches (Winkler 2005). Typically found in the 5'-UTRs of certain
bacterial mRNAs, members of each known riboswitch class form a structured
receptor (or "aptamer") (Mandal 2004) that has evolved to bind a specific
fundamental metabolite. In most cases, ligand binding regulates the
expression of a gene or group of genes involved in the synthesis or
transport of the bound metabolite. Because the biochemical pathways
regulated by riboswitches are often essential for bacterial survival,
repression of these pathways through riboswitch targeting can be lethal.

[0075]Phylogenetic sequence comparison and structural probing data
revealed that, when bound to lysine, the receptor domain of a lysine
riboswitch forms a secondary structure comprised of five stem-loops (P1
through P5) that radiate from a highly conserved single-stranded core
(FIG. 1a; Supplementary FIG. 1) (Sudarsan 2003; Grundy 2003; Rodionov
2003). The terminal loops of stems P2 and P3 base pair with one another,
and P2 also contains both a loop-E structural motif (Wimberly 1993) and a
K-turn motif (Klein 2001). In most bacteria, stabilization of this
structure by lysine binding permits the formation of a transcription
terminator that halts RNA synthesis before the downstream open reading
frame (ORF) can be transcribed (Sudarsan 2003). At subsaturating lysine
concentrations, the riboswitch forms an alternate structure in which an
antiterminator hairpin (FIG. 1a) precludes formation of the terminator
hairpin, thus enabling normal transcription of the adjoining ORF.

[0076]In many bacteria, including some clinically relevant pathogens
(Table 1), a lysine riboswitch regulates the expression of aspartokinase
II (coded by the lysC gene in Bacillus subtilis, FIG. 1b), which
catalyzes the first step in lysine, threonine, and methionine
biosynthesis (FIG. 6) (Sudarsan 2003; Grundy 2003; Rodionov 2003). Two
lysine intermediates downstream of
aspartate-4-phosphate--2,3-dihydropicolinate and L,L-diaminopimelate--are
also precursors for cell wall biosynthesis and spore formation (Hutton
2003; Bugg 1994). Many bacteria also have a second copy of the riboswitch
that regulates the expression of a lysine-specific importer (coded by the
yvsH gene in B. subtilis) (Rodionov 2003). Compounds are disclosed herein
that bind to the lysine riboswitch receptor and inhibit growth by
repressing these genes, even when the bacterium is starved for lysine.

[0078]It is to be understood that the disclosed method and compositions
are not limited to specific synthetic methods, specific analytical
techniques, or to particular reagents unless otherwise specified, and, as
such, can vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only and
is not intended to be limiting.

Materials

[0079]Disclosed are materials, compositions, and components that can be
used for, can be used in conjunction with, can be used in preparation
for, or are products of the disclosed methods and compositions. These and
other materials are disclosed herein, and it is understood that when
combinations, subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference to each of various individual and
collective combinations and permutation of these compounds can not be
explicitly disclosed, each is specifically contemplated and described
herein. For example, if a riboswitch or aptamer domain is disclosed and
discussed and a number of modifications that can be made to a number of
molecules including the riboswitch or aptamer domain are discussed, each
and every combination and permutation of riboswitch or aptamer domain and
the modifications that are possible are specifically contemplated unless
specifically indicated to the contrary. Thus, if a class of molecules A,
B, and C are disclosed as well as a class of molecules D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if each is
not individually recited, each is individually and collectively
contemplated. Thus, in this example, each of the combinations A-E, A-F,
B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should
be considered disclosed from disclosure of A, B, and C; D, E, and F; and
the example combination A-D. Likewise, any subset or combination of these
is also specifically contemplated and disclosed. Thus, for example, the
sub-group of A-E, B-F, and C-E are specifically contemplated and should
be considered disclosed from disclosure of A, B, and C; D, E, and F; and
the example combination A-D. This concept applies to all aspects of this
application including, but not limited to, steps in methods of making and
using the disclosed compositions. Thus, if there are a variety of
additional steps that can be performed it is understood that each of
these additional steps can be performed with any specific embodiment or
combination of embodiments of the disclosed methods, and that each such
combination is specifically contemplated and should be considered
disclosed.

A. Riboswitches

[0080]Riboswitches are expression control elements that are part of an RNA
molecule to be expressed and that change state when bound by a trigger
molecule. Riboswitches typically can be dissected into two separate
domains: one that selectively binds the target (aptamer domain) and
another that influences genetic control (expression platform domain). It
is the dynamic interplay between these two domains that results in
metabolite-dependent allosteric control of gene expression. Disclosed are
isolated and recombinant riboswitches, recombinant constructs containing
such riboswitches, heterologous sequences operably linked to such
riboswitches, and cells and transgenic organisms harboring such
riboswitches, riboswitch recombinant constructs, and riboswitches
operably linked to heterologous sequences. The heterologous sequences can
be, for example, sequences encoding proteins or peptides of interest,
including reporter proteins or peptides. Preferred riboswitches are, or
are derived from, naturally occurring riboswitches.

[0081]The disclosed riboswitches, including the derivatives and
recombinant forms thereof, generally can be from any source, including
naturally occurring riboswitches and riboswitches designed de novo. Any
such riboswitches can be used in or with the disclosed methods. However,
different types of riboswitches can be defined and some such sub-types
can be useful in or with particular methods (generally as described
elsewhere herein). Types of riboswitches include, for example, naturally
occurring riboswitches, derivatives and modified forms of naturally
occurring riboswitches, chimeric riboswitches, and recombinant
riboswitches. A naturally occurring riboswitch is a riboswitch having the
sequence of a riboswitch as found in nature. Such a naturally occurring
riboswitch can be an isolated or recombinant form of the naturally
occurring riboswitch as it occurs in nature. That is, the riboswitch has
the same primary structure but has been isolated or engineered in a new
genetic or nucleic acid context. Chimeric riboswitches can be made up of,
for example, part of a riboswitch of any or of a particular class or type
of riboswitch and part of a different riboswitch of the same or of any
different class or type of riboswitch; part of a riboswitch of any or of
a particular class or type of riboswitch and any non-riboswitch sequence
or component. Recombinant riboswitches are riboswitches that have been
isolated or engineered in a new genetic or nucleic acid context.

[0082]Riboswitches can have single or multiple aptamer domains. Aptamer
domains in riboswitches having multiple aptamer domains can exhibit
cooperative binding of trigger molecules or can not exhibit cooperative
binding of trigger molecules (that is, the aptamers need not exhibit
cooperative binding). In the latter case, the aptamer domains can be said
to be independent binders. Riboswitches having multiple aptamers can have
one or multiple expression platform domains. For example, a riboswitch
having two aptamer domains that exhibit cooperative binding of their
trigger molecules can be linked to a single expression platform domain
that is regulated by both aptamer domains. Riboswitches having multiple
aptamers can have one or more of the aptamers joined via a linker. Where
such aptamers exhibit cooperative binding of trigger molecules, the
linker can be a cooperative linker.

[0083]Aptamer domains can be said to exhibit cooperative binding if they
have a Hill coefficient n between x and x-1, where x is the number of
aptamer domains (or the number of binding sites on the aptamer domains)
that are being analyzed for cooperative binding. Thus, for example, a
riboswitch having two aptamer domains (such as glycine-responsive
riboswitches) can be said to exhibit cooperative binding if the
riboswitch has Hill coefficient between 2 and 1. It should be understood
that the value of x used depends on the number of aptamer domains being
analyzed for cooperative binding, not necessarily the number of aptamer
domains present in the riboswitch. This makes sense because a riboswitch
can have multiple aptamer domains where only some exhibit cooperative
binding.

[0084]Disclosed are chimeric riboswitches containing heterologous aptamer
domains and expression platform domains. That is, chimeric riboswitches
are made up an aptamer domain from one source and an expression platform
domain from another source. The heterologous sources can be from, for
example, different specific riboswitches, different types of
riboswitches, or different classes of riboswitches. The heterologous
aptamers can also come from non-riboswitch aptamers. The heterologous
expression platform domains can also come from non-riboswitch sources.

[0085]Modified or derivative riboswitches can be produced using in vitro
selection and evolution techniques. In general, in vitro evolution
techniques as applied to riboswitches involve producing a set of variant
riboswitches where part(s) of the riboswitch sequence is varied while
other parts of the riboswitch are held constant. Activation, deactivation
or blocking (or other functional or structural criteria) of the set of
variant riboswitches can then be assessed and those variant riboswitches
meeting the criteria of interest are selected for use or further rounds
of evolution. Useful base riboswitches for generation of variants are the
specific and consensus riboswitches disclosed herein. Consensus
riboswitches can be used to inform which part(s) of a riboswitch to vary
for in vitro selection and evolution.

[0086]Also disclosed are modified riboswitches with altered regulation.
The regulation of a riboswitch can be altered by operably linking an
aptamer domain to the expression platform domain of the riboswitch (which
is a chimeric riboswitch). The aptamer domain can then mediate regulation
of the riboswitch through the action of, for example, a trigger molecule
for the aptamer domain. Aptamer domains can be operably linked to
expression platform domains of riboswitches in any suitable manner,
including, for example, by replacing the normal or natural aptamer domain
of the riboswitch with the new aptamer domain. Generally, any compound or
condition that can activate, deactivate or block the riboswitch from
which the aptamer domain is derived can be used to activate, deactivate
or block the chimeric riboswitch.

[0087]Also disclosed are inactivated riboswitches. Riboswitches can be
inactivated by covalently altering the riboswitch (by, for example,
crosslinking parts of the riboswitch or coupling a compound to the
riboswitch). Inactivation of a riboswitch in this manner can result from,
for example, an alteration that prevents the trigger molecule for the
riboswitch from binding, that prevents the change in state of the
riboswitch upon binding of the trigger molecule, or that prevents the
expression platform domain of the riboswitch from affecting expression
upon binding of the trigger molecule.

[0088]Also disclosed are biosensor riboswitches. Biosensor riboswitches
are engineered riboswitches that produce a detectable signal in the
presence of their cognate trigger molecule. Useful biosensor riboswitches
can be triggered at or above threshold levels of the trigger molecules.
Biosensor riboswitches can be designed for use in vivo or in vitro. For
example, biosensor riboswitches operably linked to a reporter RNA that
encodes a protein that serves as or is involved in producing a signal can
be used in vivo by engineering a cell or organism to harbor a nucleic
acid construct encoding the riboswitch/reporter RNA. An example of a
biosensor riboswitch for use in vitro is a riboswitch that includes a
conformation dependent label, the signal from which changes depending on
the activation state of the riboswitch. Such a biosensor riboswitch
preferably uses an aptamer domain from or derived from a naturally
occurring riboswitch. Biosensor riboswitches can be used in various
situations and platforms. For example, biosensor riboswitches can be used
with solid supports, such as plates, chips, strips and wells.

[0089]Also disclosed are modified or derivative riboswitches that
recognize new trigger molecules. New riboswitches and/or new aptamers
that recognize new trigger molecules can be selected for, designed or
derived from known riboswitches. This can be accomplished by, for
example, producing a set of aptamer variants in a riboswitch, assessing
the activation of the variant riboswitches in the presence of a compound
of interest, selecting variant riboswitches that were activated (or, for
example, the riboswitches that were the most highly or the most
selectively activated), and repeating these steps until a variant
riboswitch of a desired activity, specificity, combination of activity
and specificity, or other combination of properties results.

[0090]In general, any aptamer domain can be adapted for use with any
expression platform domain by designing or adapting a regulated strand in
the expression platform domain to be complementary to the control strand
of the aptamer domain. Alternatively, the sequence of the aptamer and
control strands of an aptamer domain can be adapted so that the control
strand is complementary to a functionally significant sequence in an
expression platform. For example, the control strand can be adapted to be
complementary to the Shine-Dalgarno sequence of an RNA such that, upon
formation of a stem structure between the control strand and the SD
sequence, the SD sequence becomes inaccessible to ribosomes, thus
reducing or preventing translation initiation. Note that the aptamer
strand would have corresponding changes in sequence to allow formation of
a P1 stem in the aptamer domain. In the case of riboswitches having
multiple aptamers exhibiting cooperative binding, one the P1 stem of the
activating aptamer (the aptamer that interacts with the expression
platform domain) need be designed to form a stem structure with the SD
sequence.

[0091]As another example, a transcription terminator can be added to an
RNA molecule (most conveniently in an untranslated region of the RNA)
where part of the sequence of the transcription terminator is
complementary to the control strand of an aptamer domain (the sequence
will be the regulated strand). This will allow the control sequence of
the aptamer domain to form alternative stem structures with the aptamer
strand and the regulated strand, thus either forming or disrupting a
transcription terminator stem upon activation or deactivation of the
riboswitch. Any other expression element can be brought under the control
of a riboswitch by similar design of alternative stem structures.

[0092]For transcription terminators controlled by riboswitches, the speed
of transcription and spacing of the riboswitch and expression platform
elements can be important for proper control. Transcription speed can be
adjusted by, for example, including polymerase pausing elements (e.g., a
series of uridine residues) to pause transcription and allow the
riboswitch to form and sense trigger molecules.

[0093]Disclosed are regulatable gene expression constructs comprising a
nucleic acid molecule encoding an RNA comprising a riboswitch operably
linked to a coding region, wherein the riboswitch regulates expression of
the RNA, wherein the riboswitch and coding region are heterologous. The
riboswitch can comprise an aptamer domain and an expression platform
domain, wherein the aptamer domain and the expression platform domain are
heterologous. The riboswitch can comprise an aptamer domain and an
expression platform domain, wherein the aptamer domain comprises a P1
stem, wherein the P1 stem comprises an aptamer strand and a control
strand, wherein the expression platform domain comprises a regulated
strand, wherein the regulated strand, the control strand, or both have
been designed to form a stem structure. The riboswitch can comprise two
or more aptamer domains and an expression platform domain, wherein at
least one of the aptamer domains and the expression platform domain are
heterologous. The riboswitch can comprise two or more aptamer domains and
an expression platform domain, wherein at least one of the aptamer
domains comprises a P1 stem, wherein the P1 stem comprises an aptamer
strand and a control strand, wherein the expression platform domain
comprises a regulated strand, wherein the regulated strand, the control
strand, or both have been designed to form a stem structure.

[0094]1. Aptamer Domains

[0095]Aptamers are nucleic acid segments and structures that can bind
selectively to particular compounds and classes of compounds.
Riboswitches have aptamer domains that, upon binding of a trigger
molecule result in a change in the state or structure of the riboswitch.
In functional riboswitches, the state or structure of the expression
platform domain linked to the aptamer domain changes when the trigger
molecule binds to the aptamer domain. Aptamer domains of riboswitches can
be derived from any source, including, for example, natural aptamer
domains of riboswitches, artificial aptamers, engineered, selected,
evolved or derived aptamers or aptamer domains. Aptamers in riboswitches
generally have at least one portion that can interact, such as by forming
a stem structure, with a portion of the linked expression platform
domain. This stem structure will either form or be disrupted upon binding
of the trigger molecule.

[0096]Consensus aptamer domains of a variety of natural riboswitches are
shown in FIG. 11 of U.S. Application Publication No. 2005-0053951 and
elsewhere herein. The consensus sequence and structure for the lysine
ribozyme can be found in FIG. 5, and an example of the structure of a
lysine riboswitch can be found in FIG. 1. These aptamer domains
(including all of the direct variants embodied therein) can be used in
riboswitches. The consensus sequences and structures indicate variations
in sequence and structure. Aptamer domains that are within the indicated
variations are referred to herein as direct variants. These aptamer
domains can be modified to produce modified or variant aptamer domains.
Conservative modifications include any change in base paired nucleotides
such that the nucleotides in the pair remain complementary. Moderate
modifications include changes in the length of stems or of loops (for
which a length or length range is indicated) of less than or equal to 20%
of the length range indicated. Loop and stem lengths are considered to be
"indicated" where the consensus structure shows a stem or loop of a
particular length or where a range of lengths is listed or depicted.
Moderate modifications include changes in the length of stems or of loops
(for which a length or length range is not indicated) of less than or
equal to 40% of the length range indicated. Moderate modifications also
include and functional variants of unspecified portions of the aptamer
domain.

[0097]The P1 stem and its constituent strands can be modified in adapting
aptamer domains for use with expression platforms and RNA molecules. Such
modifications, which can be extensive, are referred to herein as P1
modifications. P1 modifications include changes to the sequence and/or
length of the P1 stem of an aptamer domain. The aptamer domain is
particularly useful as initial sequences for producing derived aptamer
domains via in vitro selection or in vitro evolution techniques.

[0098]Aptamer domains of the disclosed riboswitches can also be used for
any other purpose, and in any other context, as aptamers. For example,
aptamers can be used to control ribozymes, other molecular switches, and
any RNA molecule where a change in structure can affect function of the
RNA.

[0099]2. Expression Platform Domains

[0100]Expression platform domains are a part of riboswitches that affect
expression of the RNA molecule that contains the riboswitch. Expression
platform domains generally have at least one portion that can interact,
such as by forming a stem structure, with a portion of the linked aptamer
domain. This stem structure will either form or be disrupted upon binding
of the trigger molecule. The stem structure generally either is, or
prevents formation of, an expression regulatory structure. An expression
regulatory structure is a structure that allows, prevents, enhances or
inhibits expression of an RNA molecule containing the structure. Examples
include Shine-Dalgarno sequences, initiation codons, transcription
terminators, and stability and processing signals.

B. Trigger Molecules

[0101]Trigger molecules are molecules and compounds that can activate a
riboswitch. This includes the natural or normal trigger molecule for the
riboswitch and other compounds that can activate the riboswitch. Natural
or normal trigger molecules are the trigger molecule for a given
riboswitch in nature or, in the case of some non-natural riboswitches,
the trigger molecule for which the riboswitch was designed or with which
the riboswitch was selected (as in, for example, in vitro selection or in
vitro evolution techniques).

C. Compounds

[0102]Also disclosed are compounds, and compositions containing such
compounds, that can activate, deactivate or block a riboswitch.
Riboswitches function to control gene expression through the binding or
removal of a trigger molecule. Compounds can be used to activate,
deactivate or block a riboswitch. The trigger molecule for a riboswitch
(as well as other activating compounds) can be used to activate a
riboswitch. Compounds other than the trigger molecule generally can be
used to deactivate or block a riboswitch. Riboswitches can also be
deactivated by, for example, removing trigger molecules from the presence
of the riboswitch. A riboswitch can be blocked by, for example, binding
of an analog of the trigger molecule that does not activate the
riboswitch.

[0103]Also disclosed are compounds for altering expression of an RNA
molecule, or of a gene encoding an RNA molecule, where the RNA molecule
includes a riboswitch. This can be accomplished by bringing a compound
into contact with the RNA molecule. Riboswitches function to control gene
expression through the binding or removal of a trigger molecule. Thus,
subjecting an RNA molecule of interest that includes a riboswitch to
conditions that activate, deactivate or block the riboswitch can be used
to alter expression of the RNA. Expression can be altered as a result of,
for example, termination of transcription or blocking of ribosome binding
to the RNA. Binding of a trigger molecule can, depending on the nature of
the riboswitch, reduce or prevent expression of the RNA molecule or
promote or increase expression of the RNA molecule.

[0104]Also disclosed are compounds for regulating expression of an RNA
molecule, or of a gene encoding an RNA molecule. Also disclosed are
compounds for regulating expression of a naturally occurring gene or RNA
that contains a riboswitch by activating, deactivating or blocking the
riboswitch. If the gene is essential for survival of a cell or organism
that harbors it, activating, deactivating or blocking the riboswitch can
in death, stasis or debilitation of the cell or organism.

[0105]Also disclosed are compounds for regulating expression of an
isolated, engineered or recombinant gene or RNA that contains a
riboswitch by activating, deactivating or blocking the riboswitch. If the
gene encodes a desired expression product, activating or deactivating the
riboswitch can be used to induce expression of the gene and thus result
in production of the expression product. If the gene encodes an inducer
or repressor of gene expression or of another cellular process,
activation, deactivation or blocking of the riboswitch can result in
induction, repression, or de-repression of other, regulated genes or
cellular processes. Many such secondary regulatory effects are known and
can be adapted for use with riboswitches. An advantage of riboswitches as
the primary control for such regulation is that riboswitch trigger
molecules can be small, non-antigenic molecules.

[0106]Also disclosed are methods of identifying compounds that activate,
deactivate or block a riboswitch. For example, compounds that activate a
riboswitch can be identified by bringing into contact a test compound and
a riboswitch and assessing activation of the riboswitch. If the
riboswitch is activated, the test compound is identified as a compound
that activates the riboswitch. Activation of a riboswitch can be assessed
in any suitable manner. For example, the riboswitch can be linked to a
reporter RNA and expression, expression level, or change in expression
level of the reporter RNA can be measured in the presence and absence of
the test compound. As another example, the riboswitch can include a
conformation dependent label, the signal from which changes depending on
the activation state of the riboswitch. Such a riboswitch preferably uses
an aptamer domain from or derived from a naturally occurring riboswitch.
As can be seen, assessment of activation of a riboswitch can be performed
with the use of a control assay or measurement or without the use of a
control assay or measurement. Methods for identifying compounds that
deactivate a riboswitch can be performed in analogous ways.

[0107]Identification of compounds that block a riboswitch can be
accomplished in any suitable manner. For example, an assay can be
performed for assessing activation or deactivation of a riboswitch in the
presence of a compound known to activate or deactivate the riboswitch and
in the presence of a test compound. If activation or deactivation is not
observed as would be observed in the absence of the test compound, then
the test compound is identified as a compound that blocks activation or
deactivation of the riboswitch.

[0108]Disclosed herein are analogs that interact with the lysine
riboswitch. Examples of such analogs can be found in FIG. 2. Many of the
compounds synthesized and tested bind the lysine riboswitch with
constants that are equal to that of lysine. The fact that appendages with
highly variable chemical composition exhibit function shows that numerous
variations of these chemical scaffolds can be generated and tested for
function in vitro and inside cells. Specifically, further modified
versions of these compounds can have improved binding to the lysine
riboswitch by making new contacts to other functional groups in the RNA
structure. Furthermore, modulation of bioavailability, toxicity, and
synthetic ease (among other characteristics) can be tunable by making
modifications in these two regions of the scaffold, as the structural
model for the riboswitch shows many modifications are possible at these
sites.

[0109]High-throughput screening can also be used to reveal entirely new
chemical scaffolds that also bind to riboswitch RNAs either with standard
or non-standard modes of molecular recognition. Since riboswitches are
the first major form of natural metabolite-binding RNAs to be discovered,
there has been little effort made previously to create binding assays
that can be adapted for high-throughput screening. Multiple different
approaches can be used to detect metabolite binding RNAs, including
allosteric ribozyme assays using gel-based and chip-based detection
methods, and in-line probing assays. Also disclosed are compounds made by
identifying a compound that activates, deactivates or blocks a riboswitch
and manufacturing the identified compound. This can be accomplished by,
for example, combining compound identification methods as disclosed
elsewhere herein with methods for manufacturing the identified compounds.
For example, compounds can be made by bringing into contact a test
compound and a riboswitch, assessing activation of the riboswitch, and,
if the riboswitch is activated by the test compound, manufacturing the
test compound that activates the riboswitch as the compound.

[0110]Also disclosed are compounds made by checking activation,
deactivation or blocking of a riboswitch by a compound and manufacturing
the checked compound. This can be accomplished by, for example, combining
compound activation, deactivation or blocking assessment methods as
disclosed elsewhere herein with methods for manufacturing the checked
compounds. For example, compounds can be made by bringing into contact a
test compound and a riboswitch, assessing activation of the riboswitch,
and, if the riboswitch is activated by the test compound, manufacturing
the test compound that activates the riboswitch as the compound. Checking
compounds for their ability to activate, deactivate or block a riboswitch
refers to both identification of compounds previously unknown to
activate, deactivate or block a riboswitch and to assessing the ability
of a compound to activate, deactivate or block a riboswitch where the
compound was already known to activate, deactivate or block the
riboswitch.

[0111]As used herein, the term "substituted" is contemplated to include
all permissible substituents of organic compounds. In a broad aspect, the
permissible substituents include acyclic and cyclic, branched and
unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic
substituents of organic compounds. Illustrative substituents include, for
example, those described below. The permissible substituents can be one
or more and the same or different for appropriate organic compounds. For
the purposes of this disclosure, the heteroatoms, such as nitrogen, can
have hydrogen substituents and/or any permissible substituents of organic
compounds described herein which satisfy the valences of the heteroatoms.
This disclosure is not intended to be limited in any manner by the
permissible substituents of organic compounds. Also, the terms
"substitution" or "substituted with" include the implicit proviso that
such substitution is in accordance with permitted valence of the
substituted atom and the substituent, and that the substitution results
in a stable compound, e.g., a compound that does not spontaneously
undergo transformation such as by rearrangement, cyclization,
elimination, etc.

[0112]"A1," "A2," "A3," and "A4" are used herein as
generic symbols to represent various specific substituents. These symbols
can be any substituent, not limited to those disclosed herein, and when
they are defined to be certain substituents in one instance, they can, in
another instance, be defined as some other substituents.

[0113]The term "alkyl" as used herein is a branched or unbranched
saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,
heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl,
tetracosyl, and the like. The alkyl group can also be substituted or
unsubstituted. The alkyl group can be substituted with one or more groups
including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, sulfo-oxo, sulfonyl, sulfone, sulfoxide,
or thiol, as described below. The term "lower alkyl" is an alkyl group
with 6 or fewer carbon atoms, e.g., methyl, ethyl, propyl, isopropyl,
butyl, sec-butyl, iso-butyl, tert-butyl, pentyl, hexyl, and the like.

[0114]Throughout the specification "alkyl" is generally used to refer to
both unsubstituted alkyl groups and substituted alkyl groups; however,
substituted alkyl groups are also specifically referred to herein by
identifying the specific substituent(s) on the alkyl group. For example,
the term "halogenated alkyl" specifically refers to an alkyl group that
is substituted with one or more halide, e.g., fluorine, chlorine,
bromine, or iodine. The term "alkoxyalkyl" specifically refers to an
alkyl group that is substituted with one or more alkoxy groups, as
described below. The term "alkylamino" specifically refers to an alkyl
group that is substituted with one or more amino groups, as described
below, and the like. When "alkyl" is used in one instance and a specific
term such as "halogenated alkyl" is used in another, it is not meant to
imply that the term "alkyl" does not also refer to specific terms such as
"halogenated alkyl" and the like.

[0115]This practice is also used for other groups described herein. That
is, while a term such as "cycloalkyl" refers to both unsubstituted and
substituted cycloalkyl moieties, the substituted moieties can, in
addition, be specifically identified herein; for example, a particular
substituted cycloalkyl can be referred to as, e.g., an "alkylcycloalkyl."
Similarly, a substituted alkoxy can be specifically referred to as, e.g.,
a "halogenated alkoxy," a particular substituted alkenyl can be, e.g., an
"alkenylalcohol," and the like. Again, the practice of using a general
term, such as "cycloalkyl," and a specific term, such as
"alkylcycloalkyl," is not meant to imply that the general term does not
also include the specific term.

[0116]The term "alkoxy" as used herein is an alkyl group bonded through a
single, terminal ether linkage; that is, an "alkoxy" group can be defined
as --OA1 where A2 is alkyl as defined above.

[0117]The term "alkenyl" as used herein is a hydrocarbon group of from 2
to 24 carbon atoms with a structural formula containing at least one
carbon-carbon double bond. Asymmetric structures such as
(A'A2)C═C(A3A4) are intended to include both the E and
Z isomers. This can be presumed in structural formulae herein wherein an
asymmetric alkene is present, or it can be explicitly indicated by the
bond symbol C═C. The alkenyl group can be substituted with one or
more groups including, but not limited to, alkyl, halogenated alkyl,
alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic
acid, ester, ether, halide, hydroxy, ketone, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol, as described below.

[0118]The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24
carbon atoms with a structural formula containing at least one
carbon-carbon triple bond. The alkynyl group can be substituted with one
or more groups including, but not limited to, alkyl, halogenated alkyl,
alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic
acid, ester, ether, halide, hydroxy, ketone, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol, as described below.

[0119]The term "aryl" as used herein is a group that contains any
carbon-based aromatic group including, but not limited to, benzene,
naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term
"aryl" also includes "heteroaryl," which is defined as a group that
contains an aromatic group that has at least one heteroatom incorporated
within the ring of the aromatic group. Examples of heteroatoms include,
but are not limited to, nitrogen, oxygen, sulfur, and phosphorus.
Likewise, the term "non-heteroaryl," which is also included in the term
"aryl," defines a group that contains an aromatic group that does not
contain a heteroatom. The aryl group can be substituted or unsubstituted.
The aryl group can be substituted with one or more groups including, but
not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as
described herein. The term "biaryl" is a specific type of aryl group and
is included in the definition of aryl. Biaryl refers to two aryl groups
that are bound together via a fused ring structure, as in naphthalene, or
are attached via one or more carbon-carbon bonds, as in biphenyl.

[0120]The term "cycloalkyl" as used herein is a non-aromatic carbon-based
ring composed of at least three carbon atoms. Examples of cycloalkyl
groups include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, etc. The term "heterocycloalkyl" is a cycloalkyl
group as defined above where at least one of the carbon atoms of the ring
is substituted with a heteroatom such as, but not limited to, nitrogen,
oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl
group can be substituted or unsubstituted. The cycloalkyl group and
heterocycloalkyl group can be substituted with one or more groups
including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as
described herein.

[0121]The term "cycloalkenyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms and containing
at least one double bound, i.e., C═C. Examples of cycloalkenyl groups
include, but are not limited to, cyclopropenyl, cyclobutenyl,
cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the
like. The term "heterocycloalkenyl" is a type of cycloalkenyl group as
defined above, and is included within the meaning of the term
"cycloalkenyl," where at least one of the carbon atoms of the ring is
substituted with a heteroatom such as, but not limited to, nitrogen,
oxygen, sulfur, or phosphorus. The cycloalkenyl group and
heterocycloalkenyl group can be substituted or unsubstituted. The
cycloalkenyl group and heterocycloalkenyl group can be substituted with
one or more groups including, but not limited to, alkyl, alkoxy, alkenyl,
alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, sulfo-oxo, sulfonyl, sulfone, sulfoxide,
or thiol as described herein.

[0122]The term "cyclic group" is used herein to refer to either aryl
groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl,
cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have
one or more ring systems that can be substituted or unsubstituted. A
cyclic group can contain one or more aryl groups, one or more non-aryl
groups, or one or more aryl groups and one or more non-aryl groups.

[0123]The term "aldehyde" as used herein is represented by the formula
--C(O)H. Throughout this specification "C(O)" is a short hand notation
for C═O.

[0124]The terms "amine" or "amino" as used herein are represented by the
formula NA1A2A3, where A1, A2, and A3 can
be, independently, hydrogen, an alkyl, halogenated alkyl, alkenyl,
alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above.

[0125]The term "carboxylic acid" as used herein is represented by the
formula --C(O)OH. A "carboxylate" as used herein is represented by the
formula --C(O)O-.

[0126]The term "ester" as used herein is represented by the formula
--OC(O)A1 or --C(O)OA1, where A1 can be an alkyl,
halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described
above.

[0127]The term "ether" as used herein is represented by the formula
A1OA2, where A1 and A2 can be, independently, an
alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described
above.

[0128]The term "ketone" as used herein is represented by the formula
A1C(O)A2, where A1 and A2 can be, independently, an
alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described
above.

[0129]The term "halide" as used herein refers to the halogens fluorine,
chlorine, bromine, and iodine.

[0130]The term "hydroxyl" as used herein is represented by the formula
--OH.

[0131]The term "sulfo-oxo" as used herein is represented by the formulas
--S(O)A1 (i.e., "sulfonyl"), A1S(O)A2 (i.e., "sulfoxide"),
--S(O)2A1, A1SO2A2 (i.e., "sulfone"),
--OS(O)2A1, or --OS(O)2OA1, where A1 and A2
can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl,
heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above. Throughout this specification
"S(O)" is a short hand notation for S═O.

[0132]The term "sulfonylamino" or "sulfonamide" as used herein is
represented by the formula --S(O)2NH--.

[0133]The term "thiol" as used herein is represented by the formula --SH.

[0134]As used herein, "Rn" where n is some integer can independently
possess one or more of the groups listed above. Depending upon the groups
that are selected, a first group can be incorporated within second group
or, alternatively, the first group can be pendant (i.e., attached) to the
second group. For example, with the phrase "an alkyl group comprising an
amino group," the amino group can be incorporated within the backbone of
the alkyl group. Alternatively, the amino group can be attached to the
backbone of the alkyl group. The nature of the group(s) that is (are)
selected will determine if the first group is embedded or attached to the
second group.

[0135]Unless stated to the contrary, a formula with chemical bonds shown
only as solid lines and not as wedges or dashed lines contemplates each
possible isomer, e.g., each enantiomer and diastereomer, and a mixture of
isomers, such as a racemic or scalemic mixture.

[0136]Certain materials, compounds, compositions, and components disclosed
herein can be obtained commercially or readily synthesized using
techniques generally known to those of skill in the art. For example, the
starting materials and reagents used in preparing the disclosed compounds
and compositions are either available from commercial suppliers such as
Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains,
N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or
are prepared by methods known to those skilled in the art following
procedures set forth in references such as Fieser and Fieser's Reagents
for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's
Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier
Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley
and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and
Sons, 4th Edition); and Larock's Comprehensive Organic Transformations
(VCH Publishers Inc., 1989).

[0146]wherein can each independently represent a single or double bond,
and

[0147]wherein the compound is not lysine.

[0148]R3 can be positively charged and can serve as a hydrogen bond
donor. R5 can be uncharged. R9 can be C, O, or S. The pKa
of R3 can be 7 or higher. R13 can be positively charged, and
can serve as a hydrogen bond donor, or both.

[0149]In one example, R6, R7, R8, R9, R10 and
R11 are not all simultaneously C, CH, or CH2.

[0150]In another example, R1, R2, R3, R4 and R9
are not simultaneously O, NH3.sup.+, NH3.sup.+, O and S,
respectively. Furthermore, in another example, R1, R2, R3,
and R4 are not simultaneously O, H, NH3.sup.+, and O,
respectively. In another example, R1, R2, R3, R4 and
R9 are not simultaneously CO2-, NH3.sup.+,
NH3.sup.+, and H, respectively. In a further example, R1,
R2, R3, R4 and R11 are not simultaneously O,
NH3.sup.+, NH3.sup.+, O and C--CO2-, respectively. In
a further example, R1, R2, R3, and R4 are not
simultaneously NHOH, NH3.sup.+, NH3.sup.+, O and S,
respectively.

[0154]In a further example, R10 can be N, NH, O, or S. In a further
example, R7 can be CH.

[0155]It is to be understood that while a particular moiety or group can
be referred to herein as a hydrogen bond donor or acceptor, this
terminology is used to merely categorize the various substituents for
ease of reference. Such language should not be interpreted to mean that a
particular moiety actually participates in hydrogen bonding with the
riboswitch or some other compound. It is possible that, for example, a
moiety referred to herein as a hydrogen bond acceptor (or donor) could
solely or additionally be involved in hydrophobic, ionic, van de Waals,
or other type of interaction with the riboswitch or other compound.

[0156]It is also understood that certain groups disclosed herein can be
referred to herein as both a hydrogen bond acceptor and a hydrogen bond
donor. For example, --OH can be a hydrogen bond donor by donating the
hydrogen atom; --OH can also be a hydrogen bond acceptor through one or
more of the nonbonded electron pairs on the oxygen atom. Thus, throughout
the specification various moieties can be a hydrogen bond donor and
acceptor and can be referred to as such.

[0157]Every compound within the above definition is intended to be and
should be considered to be specifically disclosed herein. Further, every
subgroup that can be identified within the above definition is intended
to be and should be considered to be specifically disclosed herein. As a
result, it is specifically contemplated that any compound, or subgroup of
compounds can be either specifically included for or excluded from use or
included in or excluded from a list of compounds. As an example, a group
of compounds is contemplated where each compound is as defined above and
is able to activate a lysine-responsive riboswitch.

[0158]It should be understood that particular contacts and interactions
(such as hydrogen bond donation or acceptance) described herein for
compounds interacting with riboswitches are preferred but are not
essential for interaction of a compound with a riboswitch. For example,
compounds can interact with riboswitches with less affinity and/or
specificity than compounds having the disclosed contacts and
interactions. Further, different or additional functional groups on the
compounds can introduce new, different and/or compensating contacts with
the riboswitches. For example, for lysine riboswitches, large functional
groups can be used. Such functional groups can have, and can be designed
to have, contacts and interactions with other part of the riboswitch.
Such contacts and interactions can compensate for contacts and
interactions of the trigger molecules and core structure.

D. Constructs, Vectors and Expression Systems

[0159]The disclosed lysine riboswitches can be used with any suitable
expression system. Recombinant expression is usefully accomplished using
a vector, such as a plasmid. The vector can include a promoter operably
linked to riboswitch-encoding sequence and RNA to be expression (e.g.,
RNA encoding a protein). The vector can also include other elements
required for transcription and translation. As used herein, vector refers
to any carrier containing exogenous DNA. Thus, vectors are agents that
transport the exogenous nucleic acid into a cell without degradation and
include a promoter yielding expression of the nucleic acid in the cells
into which it is delivered. Vectors include but are not limited to
plasmids, viral nucleic acids, viruses, phage nucleic acids, phages,
cosmids, and artificial chromosomes. A variety of prokaryotic and
eukaryotic expression vectors suitable for carrying riboswitch-regulated
constructs can be produced. Such expression vectors include, for example,
pET, pET3d, pCR2.1, pBAD, pUC, and yeast vectors. The vectors can be
used, for example, in a variety of in vivo and in vitro situation.

[0160]Viral vectors include adenovirus, adeno-associated virus, herpes
virus, vaccinia virus, polio virus, AIDS virus, neuronal trophic virus,
Sindbis and other RNA viruses, including these viruses with the HIV
backbone. Also useful are any viral families which share the properties
of these viruses which make them suitable for use as vectors. Retroviral
vectors, which are described in Verma (1985), include Murine Maloney
Leukemia virus, MMLV, and retroviruses that express the desirable
properties of MMLV as a vector. Typically, viral vectors contain,
nonstructural early genes, structural late genes, an RNA polymerase III
transcript, inverted terminal repeats necessary for replication and
encapsidation, and promoters to control the transcription and replication
of the viral genome. When engineered as vectors, viruses typically have
one or more of the early genes removed and a gene or gene/promoter
cassette is inserted into the viral genome in place of the removed viral
DNA.

[0161]A "promoter" is generally a sequence or sequences of DNA that
function when in a relatively fixed location in regard to the
transcription start site. A "promoter" contains core elements required
for basic interaction of RNA polymerase and transcription factors and can
contain upstream elements and response elements.

[0162]"Enhancer" generally refers to a sequence of DNA that functions at
no fixed distance from the transcription start site and can be either 5'
(Laimins, 1981) or 3' (Lusky et al., 1983) to the transcription unit.
Furthermore, enhancers can be within an intron (Banerji et al., 1983) as
well as within the coding sequence itself (Osborne et al., 1984). They
are usually between 10 and 300 by in length, and they function in cis.
Enhancers function to increase transcription from nearby promoters.
Enhancers, like promoters, also often contain response elements that
mediate the regulation of transcription. Enhancers often determine the
regulation of expression.

[0163]Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant, animal, human or nucleated cells) can also contain
sequences necessary for the termination of transcription which can affect
mRNA expression. These regions are transcribed as polyadenylated segments
in the untranslated portion of the mRNA encoding tissue factor protein.
The 3' untranslated regions also include transcription termination sites.
It is preferred that the transcription unit also contain a
polyadenylation region. One benefit of this region is that it increases
the likelihood that the transcribed unit will be processed and
transported like mRNA. The identification and use of polyadenylation
signals in expression constructs is well established. It is preferred
that homologous polyadenylation signals be used in the transgene
constructs.

[0164]The vector can include nucleic acid sequence encoding a marker
product. This marker product is used to determine if the gene has been
delivered to the cell and once delivered is being expressed. Preferred
marker genes are the E. Coli lacZ gene which encodes β-galactosidase
and green fluorescent protein.

[0165]In some embodiments the marker can be a selectable marker. When such
selectable markers are successfully transferred into a host cell, the
transformed host cell can survive if placed under selective pressure.
There are two widely used distinct categories of selective regimes. The
first category is based on a cell's metabolism and the use of a mutant
cell line which lacks the ability to grow independent of a supplemented
media. The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use of a
mutant cell line. These schemes typically use a drug to arrest growth of
a host cell. Those cells which have a novel gene would express a protein
conveying drug resistance and would survive the selection. Examples of
such dominant selection use the drugs neomycin, (Southern and Berg,
1982), mycophenolic acid, (Mulligan and Berg, 1980) or hygromycin (Sugden
et al., 1985).

[0166]Gene transfer can be obtained using direct transfer of genetic
material, in but not limited to, plasmids, viral vectors, viral nucleic
acids, phage nucleic acids, phages, cosmids, and artificial chromosomes,
or via transfer of genetic material in cells or carriers such as cationic
liposomes. Such methods are well known in the art and readily adaptable
for use in the method described herein. Transfer vectors can be any
nucleotide construction used to deliver genes into cells (e.g., a
plasmid), or as part of a general strategy to deliver genes, e.g., as
part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res.
53:83-88, (1993)). Appropriate means for transfection, including viral
vectors, chemical transfectants, or physico-mechanical methods such as
electroporation and direct diffusion of DNA, are described by, for
example, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); and
Wolff, J. A. Nature, 352, 815-818, (1991).

[0167]1. Viral Vectors

[0168]Preferred viral vectors are Adenovirus, Adeno-associated virus,
Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic
virus, Sindbis and other RNA viruses, including these viruses with the
HIV backbone. Also preferred are any viral families which share the
properties of these viruses which make them suitable for use as vectors.
Preferred retroviruses include Murine Maloney Leukemia virus, MMLV, and
retroviruses that express the desirable properties of MMLV as a vector.
Retroviral vectors are able to carry a larger genetic payload, i.e., a
transgene or marker gene, than other viral vectors, and for this reason
are a commonly used vector. However, they are not useful in
non-proliferating cells. Adenovirus vectors are relatively stable and
easy to work with, have high titers, and can be delivered in aerosol
formulation, and can transfect non-dividing cells. Pox viral vectors are
large and have several sites for inserting genes, they are thermostable
and can be stored at room temperature. A preferred embodiment is a viral
vector which has been engineered so as to suppress the immune response of
the host organism, elicited by the viral antigens. Preferred vectors of
this type will carry coding regions for Interleukin 8 or 10.

[0169]Viral vectors have higher transaction (ability to introduce genes)
abilities than do most chemical or physical methods to introduce genes
into cells. Typically, viral vectors contain, nonstructural early genes,
structural late genes, an RNA polymerase III transcript, inverted
terminal repeats necessary for replication and encapsidation, and
promoters to control the transcription and replication of the viral
genome. When engineered as vectors, viruses typically have one or more of
the early genes removed and a gene or gene/promoter cassette is inserted
into the viral genome in place of the removed viral DNA. Constructs of
this type can carry up to about 8 kb of foreign genetic material. The
necessary functions of the removed early genes are typically supplied by
cell lines which have been engineered to express the gene products of the
early genes in trans.

[0170]i. Retroviral Vectors

[0171]A retrovirus is an animal virus belonging to the virus family of
Retroviridae, including any types, subfamilies, genus, or tropisms.
Retroviral vectors, in general, are described by Verma, I. M., Retroviral
vectors for gene transfer. In Microbiology-1985, American Society for
Microbiology, pp. 229-232, Washington, (1985), which is incorporated by
reference herein. Examples of methods for using retroviral vectors for
gene therapy are described in U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT
applications WO 90/02806 and WO 89/07136; and Mulligan, (Science
260:926-932 (1993)); the teachings of which are incorporated herein by
reference.

[0172]A retrovirus is essentially a package which has packed into it
nucleic acid cargo. The nucleic acid cargo carries with it a packaging
signal, which ensures that the replicated daughter molecules will be
efficiently packaged within the package coat. In addition to the package
signal, there are a number of molecules which are needed in cis, for the
replication, and packaging of the replicated virus. Typically a
retroviral genome, contains the gag, pol, and env genes which are
involved in the making of the protein coat. It is the gag, pol, and env
genes which are typically replaced by the foreign DNA that it is to be
transferred to the target cell. Retrovirus vectors typically contain a
packaging signal for incorporation into the package coat, a sequence
which signals the start of the gag transcription unit, elements necessary
for reverse transcription, including a primer binding site to bind the
tRNA primer of reverse transcription, terminal repeat sequences that
guide the switch of RNA strands during DNA synthesis, a purine rich
sequence 5' to the 3' LTR that serve as the priming site for the
synthesis of the second strand of DNA synthesis, and specific sequences
near the ends of the LTRs that enable the insertion of the DNA state of
the retrovirus to insert into the host genome. The removal of the gag,
pol, and env genes allows for about 8 kb of foreign sequence to be
inserted into the viral genome, become reverse transcribed, and upon
replication be packaged into a new retroviral particle. This amount of
nucleic acid is sufficient for the delivery of a one to many genes
depending on the size of each transcript. It is preferable to include
either positive or negative selectable markers along with other genes in
the insert.

[0173]Since the replication machinery and packaging proteins in most
retroviral vectors have been removed (gag, pol, and env), the vectors are
typically generated by placing them into a packaging cell line. A
packaging cell line is a cell line which has been transfected or
transformed with a retrovirus that contains the replication and packaging
machinery, but lacks any packaging signal. When the vector carrying the
DNA of choice is transfected into these cell lines, the vector containing
the gene of interest is replicated and packaged into new retroviral
particles, by the machinery provided in cis by the helper cell. The
genomes for the machinery are not packaged because they lack the
necessary signals.

[0176]A preferred viral vector is one based on an adenovirus which has had
the E1 gene removed and these virons are generated in a cell line such as
the human 293 cell line. In another preferred embodiment both the E1 and
E3 genes are removed from the adenovirus genome.

[0177]Another type of viral vector is based on an adeno-associated virus
(AAV). This defective parvovirus is a preferred vector because it can
infect many cell types and is nonpathogenic to humans. AAV type vectors
can transport about 4 to 5 kb and wild type AAV is known to stably insert
into chromosome 19. Vectors which contain this site specific integration
property are preferred. An especially preferred embodiment of this type
of vector is the P4.1 C vector produced by Avigen, San Francisco, Calif.,
which can contain the herpes simplex virus thymidine kinase gene, HSV-tk,
and/or a marker gene, such as the gene encoding the green fluorescent
protein, GFP.

[0178]The inserted genes in viral and retroviral usually contain
promoters, and/or enhancers to help control the expression of the desired
gene product. A promoter is generally a sequence or sequences of DNA that
function when in a relatively fixed location in regard to the
transcription start site. A promoter contains core elements required for
basic interaction of RNA polymerase and transcription factors, and can
contain upstream elements and response elements.

[0179]2. Viral Promoters and Enhancers

[0180]Preferred promoters controlling transcription from vectors in
mammalian host cells can be obtained from various sources, for example,
the genomes of viruses such as: polyoma, Simian Virus 40 (SV40),
adenovirus, retroviruses, hepatitis-B virus and most preferably
cytomegalovirus, or from heterologous mammalian promoters, e.g. beta
actin promoter. The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment which also contains
the SV40 viral origin of replication (Fiers et al., Nature, 273: 113
(1978)). The immediate early promoter of the human cytomegalovirus is
conveniently obtained as a HindIII E restriction fragment (Greenway, P.
J. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host
cell or related species also are useful herein.

[0181]Enhancer generally refers to a sequence of DNA that functions at no
fixed distance from the transcription start site and can be either 5'
(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lucky,
M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit.
Furthermore, enhancers can be within an intron (Banerji, J. L. et al.,
Cell 33: 729 (1983)) as well as within the coding sequence itself
(Osborne, T. F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually
between 10 and 300 by in length, and they function in cis. Enhancers
function to increase transcription from nearby promoters. Enhancers also
often contain response elements that mediate the regulation of
transcription. Promoters can also contain response elements that mediate
the regulation of transcription. Enhancers often determine the regulation
of expression of a gene. While many enhancer sequences are now known from
mammalian genes (globin, elastase, albumin, α-fetoprotein and
insulin), typically one will use an enhancer from a eukaryotic cell
virus. Preferred examples are the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.

[0182]The promoter and/or enhancer can be specifically activated either by
light or specific chemical events which trigger their function. Systems
can be regulated by reagents such as tetracycline and dexamethasone.
There are also ways to enhance viral vector gene expression by exposure
to irradiation, such as gamma irradiation, or alkylating chemotherapy
drugs.

[0183]It is preferred that the promoter and/or enhancer region be active
in all eukaryotic cell types. A preferred promoter of this type is the
CMV promoter (650 bases). Other preferred promoters are SV40 promoters,
cytomegalovirus (full length promoter), and retroviral vector LTF.

[0184]It has been shown that all specific regulatory elements can be
cloned and used to construct expression vectors that are selectively
expressed in specific cell types such as melanoma cells. The glial
fibrillary acetic protein (GFAP) promoter has been used to selectively
express genes in cells of glial origin.

[0185]Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant, animal, human or nucleated cells) can also contain
sequences necessary for the termination of transcription which can affect
mRNA expression. These regions are transcribed as polyadenylated segments
in the untranslated portion of the mRNA encoding tissue factor protein.
The 3' untranslated regions also include transcription termination sites.
It is preferred that the transcription unit also contain a
polyadenylation region. One benefit of this region is that it increases
the likelihood that the transcribed unit will be processed and
transported like mRNA. The identification and use of polyadenylation
signals in expression constructs is well established. It is preferred
that homologous polyadenylation signals be used in the transgene
constructs. In a preferred embodiment of the transcription unit, the
polyadenylation region is derived from the SV40 early polyadenylation
signal and consists of about 400 bases. It is also preferred that the
transcribed units contain other standard sequences alone or in
combination with the above sequences improve expression from, or
stability of, the construct.

[0186]3. Markers

[0187]The vectors can include nucleic acid sequence encoding a marker
product. This marker product is used to determine if the gene has been
delivered to the cell and once delivered is being expressed. Preferred
marker genes are the E. Coli lacZ gene which encodes β-galactosidase
and green fluorescent protein.

[0188]In some embodiments the marker can be a selectable marker. Examples
of suitable selectable markers for mammalian cells are dihydrofolate
reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,
hydromycin, and puromycin. When such selectable markers are successfully
transferred into a mammalian host cell, the transformed mammalian host
cell can survive if placed under selective pressure. There are two widely
used distinct categories of selective regimes. The first category is
based on a cell's metabolism and the use of a mutant cell line which
lacks the ability to grow independent of a supplemented media. Two
examples are: CHO DHFR- cells and mouse LTK- cells. These cells
lack the ability to grow without the addition of such nutrients as
thymidine or hypoxanthine. Because these cells lack certain genes
necessary for a complete nucleotide synthesis pathway, they cannot
survive unless the missing nucleotides are provided in a supplemented
media. An alternative to supplementing the media is to introduce an
intact DHFR or TK gene into cells lacking the respective genes, thus
altering their growth requirements. Individual cells which were not
transformed with the DHFR or TK gene will not be capable of survival in
non-supplemented media.

[0189]The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use of a
mutant cell line. These schemes typically use a drug to arrest growth of
a host cell. Those cells which would express a protein conveying drug
resistance and would survive the selection. Examples of such dominant
selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec.
Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R. C. and
Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al.,
Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial
genes under eukaryotic control to convey resistance to the appropriate
drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or
hygromycin, respectively. Others include the neomycin analog G418 and
puramycin.

E. Biosensor Riboswitches

[0190]Also disclosed are biosensor riboswitches. Biosensor riboswitches
are engineered riboswitches that produce a detectable signal in the
presence of their cognate trigger molecule. Useful biosensor riboswitches
can be triggered at or above threshold levels of the trigger molecules.
Biosensor riboswitches can be designed for use in vivo or in vitro. For
example, lysine biosensor riboswitches operably linked to a reporter RNA
that encodes a protein that serves as or is involved in producing a
signal can be used in vivo by engineering a cell or organism to harbor a
nucleic acid construct encoding the lysine riboswitch/reporter RNA. An
example of a biosensor riboswitch for use in vitro is a riboswitch that
includes a conformation dependent label, the signal from which changes
depending on the activation state of the riboswitch. Such a biosensor
riboswitch preferably uses an aptamer domain from or derived from a
naturally occurring riboswitch, such as lysine.

F. Reporter Proteins and Peptides

[0191]For assessing activation of a riboswitch, or for biosensor
riboswitches, a reporter protein or peptide can be used. The reporter
protein or peptide can be encoded by the RNA the expression of which is
regulated by the riboswitch. The examples describe the use of some
specific reporter proteins. The use of reporter proteins and peptides is
well known and can be adapted easily for use with riboswitches. The
reporter proteins can be any protein or peptide that can be detected or
that produces a detectable signal. Preferably, the presence of the
protein or peptide can be detected using standard techniques (e.g.,
radioimmunoassay, radio-labeling, immunoassay, assay for enzymatic
activity, absorbance, fluorescence, luminescence, and Western blot). More
preferably, the level of the reporter protein is easily quantifiable
using standard techniques even at low levels. Useful reporter proteins
include luciferases, green fluorescent proteins and their derivatives,
such as firefly luciferase (FL) from Photinus pyralis, and Renilla
luciferase (RL) from Renilla reniformis.

G. Conformation Dependent Labels

[0192]Conformation dependent labels refer to all labels that produce a
change in fluorescence intensity or wavelength based on a change in the
form or conformation of the molecule or compound (such as a riboswitch)
with which the label is associated. Examples of conformation dependent
labels used in the context of probes and primers include molecular
beacons, Amplifluors, FRET probes, cleavable FRET probes, TaqMan probes,
scorpion primers, fluorescent triplex oligos including but not limited to
triplex molecular beacons or triplex FRET probes, fluorescent
water-soluble conjugated polymers, PNA probes and QPNA probes. Such
labels, and, in particular, the principles of their function, can be
adapted for use with riboswitches. Several types of conformation
dependent labels are reviewed in Schweitzer and Kingsmore, Curr. Opin.
Biotech. 12:21-27 (2001).

[0193]Stem quenched labels, a form of conformation dependent labels, are
fluorescent labels positioned on a nucleic acid such that when a stem
structure forms a quenching moiety is brought into proximity such that
fluorescence from the label is quenched. When the stem is disrupted (such
as when a riboswitch containing the label is activated), the quenching
moiety is no longer in proximity to the fluorescent label and
fluorescence increases. Examples of this effect can be found in molecular
beacons, fluorescent triplex oligos, triplex molecular beacons, triplex
FRET probes, and QPNA probes, the operational principles of which can be
adapted for use with riboswitches.

[0194]Stem activated labels, a form of conformation dependent labels, are
labels or pairs of labels where fluorescence is increased or altered by
formation of a stem structure. Stem activated labels can include an
acceptor fluorescent label and a donor moiety such that, when the
acceptor and donor are in proximity (when the nucleic acid strands
containing the labels form a stem structure), fluorescence resonance
energy transfer from the donor to the acceptor causes the acceptor to
fluoresce. Stem activated labels are typically pairs of labels positioned
on nucleic acid molecules (such as riboswitches) such that the acceptor
and donor are brought into proximity when a stem structure is formed in
the nucleic acid molecule. If the donor moiety of a stem activated label
is itself a fluorescent label, it can release energy as fluorescence
(typically at a different wavelength than the fluorescence of the
acceptor) when not in proximity to an acceptor (that is, when a stem
structure is not formed). When the stem structure forms, the overall
effect would then be a reduction of donor fluorescence and an increase in
acceptor fluorescence. FRET probes are an example of the use of stem
activated labels, the operational principles of which can be adapted for
use with riboswitches.

H. Detection Labels

[0195]To aid in detection and quantitation of riboswitch activation,
deactivation or blocking, or expression of nucleic acids or protein
produced upon activation, deactivation or blocking of riboswitches,
detection labels can be incorporated into detection probes or detection
molecules or directly incorporated into expressed nucleic acids or
proteins. As used herein, a detection label is any molecule that can be
associated with nucleic acid or protein, directly or indirectly, and
which results in a measurable, detectable signal, either directly or
indirectly. Many such labels are known to those of skill in the art.
Examples of detection labels suitable for use in the disclosed method are
radioactive isotopes, fluorescent molecules, phosphorescent molecules,
enzymes, antibodies, and ligands.

[0198]Additional labels of interest include those that provide for signal
only when the probe with which they are associated is specifically bound
to a target molecule, where such labels include: "molecular beacons" as
described in Tyagi & Kramer, Nature Biotechnology (1996) 14:303 and EP 0
070 685 B1. Other labels of interest include those described in U.S. Pat.
No. 5,563,037; WO 97/17471 and WO 97/17076.

[0200]Detection labels that are incorporated into nucleic acid, such as
biotin, can be subsequently detected using sensitive methods well-known
in the art. For example, biotin can be detected using
streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is
bound to the biotin and subsequently detected by chemiluminescence of
suitable substrates (for example, chemiluminescent substrate CSPD:
disodium, 3-(4-methoxyspiro-[1,2,-dioxetane-3-2'-(5'-chloro)tricyclo
[3.3.1.13,7]decane]-4-yl)phenyl phosphate; Tropix, Inc.). Labels can
also be enzymes, such as alkaline phosphatase, soybean peroxidase,
horseradish peroxidase and polymerases, that can be detected, for
example, with chemical signal amplification or by using a substrate to
the enzyme which produces light (for example, a chemiluminescent
1,2-dioxetane substrate) or fluorescent signal.

[0201]Molecules that combine two or more of these detection labels are
also considered detection labels. Any of the known detection labels can
be used with the disclosed probes, tags, molecules and methods to label
and detect activated or deactivated riboswitches or nucleic acid or
protein produced in the disclosed methods. Methods for detecting and
measuring signals generated by detection labels are also known to those
of skill in the art. For example, radioactive isotopes can be detected by
scintillation counting or direct visualization; fluorescent molecules can
be detected with fluorescent spectrophotometers; phosphorescent molecules
can be detected with a spectrophotometer or directly visualized with a
camera; enzymes can be detected by detection or visualization of the
product of a reaction catalyzed by the enzyme; antibodies can be detected
by detecting a secondary detection label coupled to the antibody. As used
herein, detection molecules are molecules which interact with a compound
or composition to be detected and to which one or more detection labels
are coupled.

I. Sequence Similarities

[0202]It is understood that as discussed herein the use of the terms
homology and identity mean the same thing as similarity. Thus, for
example, if the use of the word homology is used between two sequences
(non-natural sequences, for example) it is understood that this is not
necessarily indicating an evolutionary relationship between these two
sequences, but rather is looking at the similarity or relatedness between
their nucleic acid sequences. Many of the methods for determining
homology between two evolutionarily related molecules are routinely
applied to any two or more nucleic acids or proteins for the purpose of
measuring sequence similarity regardless of whether they are
evolutionarily related or not.

[0203]In general, it is understood that one way to define any known
variants and derivatives or those that might arise, of the disclosed
riboswitches, aptamers, expression platforms, genes and proteins herein,
is through defining the variants and derivatives in terms of homology to
specific known sequences. This identity of particular sequences disclosed
herein is also discussed elsewhere herein. In general, variants of
riboswitches, aptamers, expression platforms, genes and proteins herein
disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, or 99 percent homology to a stated sequence or a native
sequence. Those of skill in the art readily understand how to determine
the homology of two proteins or nucleic acids, such as genes. For
example, the homology can be calculated after aligning the two sequences
so that the homology is at its highest level.

[0204]Another way of calculating homology can be performed by published
algorithms. Optimal alignment of sequences for comparison can be
conducted by the local homology algorithm of Smith and Waterman Adv.
Appl. Math. 2: 482 (1981), by the homology alignment algorithm of
Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for
similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.
85: 2444 (1988), by computerized implementations of these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection.

[0205]The same types of homology can be obtained for nucleic acids by for
example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,
Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et
al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by
reference for at least material related to nucleic acid alignment. It is
understood that any of the methods typically can be used and that in
certain instances the results of these various methods can differ, but
the skilled artisan understands if identity is found with at least one of
these methods, the sequences would be said to have the stated identity.

[0206]For example, as used herein, a sequence recited as having a
particular percent homology to another sequence refers to sequences that
have the recited homology as calculated by any one or more of the
calculation methods described above. For example, a first sequence has 80
percent homology, as defined herein, to a second sequence if the first
sequence is calculated to have 80 percent homology to the second sequence
using the Zuker calculation method even if the first sequence does not
have 80 percent homology to the second sequence as calculated by any of
the other calculation methods. As another example, a first sequence has
80 percent homology, as defined herein, to a second sequence if the first
sequence is calculated to have 80 percent homology to the second sequence
using both the Zuker calculation method and the Pearson and Lipman
calculation method even if the first sequence does not have 80 percent
homology to the second sequence as calculated by the Smith and Waterman
calculation method, the Needleman and Wunsch calculation method, the
Jaeger calculation methods, or any of the other calculation methods. As
yet another example, a first sequence has 80 percent homology, as defined
herein, to a second sequence if the first sequence is calculated to have
80 percent homology to the second sequence using each of calculation
methods (although, in practice, the different calculation methods will
often result in different calculated homology percentages).

J. Hybridization and Selective Hybridization

[0207]The term hybridization typically means a sequence driven interaction
between at least two nucleic acid molecules, such as a primer or a probe
and a riboswitch or a gene. Sequence driven interaction means an
interaction that occurs between two nucleotides or nucleotide analogs or
nucleotide derivatives in a nucleotide specific manner. For example, G
interacting with C or A interacting with T are sequence driven
interactions. Typically sequence driven interactions occur on the
Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization
of two nucleic acids is affected by a number of conditions and parameters
known to those of skill in the art. For example, the salt concentrations,
pH, and temperature of the reaction all affect whether two nucleic acid
molecules will hybridize.

[0208]Parameters for selective hybridization between two nucleic acid
molecules are well known to those of skill in the art. For example, in
some embodiments selective hybridization conditions can be defined as
stringent hybridization conditions. For example, stringency of
hybridization is controlled by both temperature and salt concentration of
either or both of the hybridization and washing steps. For example, the
conditions of hybridization to achieve selective hybridization can
involve hybridization in high ionic strength solution (6×SSC or
6×SSPE) at a temperature that is about 12-25° C. below the
Tm (the melting temperature at which half of the molecules dissociate
from their hybridization partners) followed by washing at a combination
of temperature and salt concentration chosen so that the washing
temperature is about 5° C. to 20° C. below the Tm. The
temperature and salt conditions are readily determined empirically in
preliminary experiments in which samples of reference DNA immobilized on
filters are hybridized to a labeled nucleic acid of interest and then
washed under conditions of different stringencies. Hybridization
temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations.
The conditions can be used as described above to achieve stringency, or
as is known in the art (Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein
incorporated by reference for material at least related to hybridization
of nucleic acids). A preferable stringent hybridization condition for a
DNA:DNA hybridization can be at about 68° C. (in aqueous solution)
in 6×SSC or 6×SSPE followed by washing at 68° C.
Stringency of hybridization and washing, if desired, can be reduced
accordingly as the degree of complementarity desired is decreased, and
further, depending upon the G-C or A-T richness of any area wherein
variability is searched for. Likewise, stringency of hybridization and
washing, if desired, can be increased accordingly as homology desired is
increased, and further, depending upon the G-C or A-T richness of any
area wherein high homology is desired, all as known in the art.

[0209]Another way to define selective hybridization is by looking at the
amount (percentage) of one of the nucleic acids bound to the other
nucleic acid. For example, in some embodiments selective hybridization
conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound
to the non-limiting nucleic acid. Typically, the non-limiting nucleic
acid is in for example, 10 or 100 or 1000 fold excess. This type of assay
can be performed at under conditions where both the limiting and
non-limiting nucleic acids are for example, 10 fold or 100 fold or 1000
fold below their kd, or where only one of the nucleic acid molecules
is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid
molecules are above their kd.

[0211]Just as with homology, it is understood that there are a variety of
methods herein disclosed for determining the level of hybridization
between two nucleic acid molecules. It is understood that these methods
and conditions can provide different percentages of hybridization between
two nucleic acid molecules, but unless otherwise indicated meeting the
parameters of any of the methods would be sufficient. For example if 80%
hybridization was required and as long as hybridization occurs within the
required parameters in any one of these methods it is considered
disclosed herein.

[0212]It is understood that those of skill in the art understand that if a
composition or method meets any one of these criteria for determining
hybridization either collectively or singly it is a composition or method
that is disclosed herein.

K. Nucleic Acids

[0213]There are a variety of molecules disclosed herein that are nucleic
acid based, including, for example, riboswitches, aptamers, and nucleic
acids that encode riboswitches and aptamers. The disclosed nucleic acids
can be made up of for example, nucleotides, nucleotide analogs, or
nucleotide substitutes. Non-limiting examples of these and other
molecules are discussed herein. It is understood that for example, when a
vector is expressed in a cell, that the expressed mRNA will typically be
made up of A, C, G, and U. Likewise, it is understood that if a nucleic
acid molecule is introduced into a cell or cell environment through for
example exogenous delivery, it is advantageous that the nucleic acid
molecule be made up of nucleotide analogs that reduce the degradation of
the nucleic acid molecule in the cellular environment.

[0214]So long as their relevant function is maintained, riboswitches,
aptamers, expression platforms and any other oligonucleotides and nucleic
acids can be made up of or include modified nucleotides (nucleotide
analogs). Many modified nucleotides are known and can be used in
oligonucleotides and nucleic acids. A nucleotide analog is a nucleotide
which contains some type of modification to either the base, sugar, or
phosphate moieties. Modifications to the base moiety would include
natural and synthetic modifications of A, C, G, and T/U as well as
different purine or pyrimidine bases, such as uracil-5-yl,
hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. A modified base includes
but is not limited to 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other
alkyl derivatives of adenine and guanine, 2-propyl and other alkyl
derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Additional base modifications can be found for example in U.S. Pat. No.
3,687,808, Englisch et al., Angewandte Chemie, International Edition,
1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press,
1993. Certain nucleotide analogs, such as 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including
2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
5-methylcytosine can increase the stability of duplex formation. Other
modified bases are those that function as universal bases. Universal
bases include 3-nitropyrrole and 5-nitroindole. Universal bases
substitute for the normal bases but have no bias in base pairing. That
is, universal bases can base pair with any other base. Base modifications
often can be combined with for example a sugar modification, such as
2'-O-methoxyethyl, to achieve unique properties such as increased duplex
stability. There are numerous United States patents such as U.S. Pat.
Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, which detail
and describe a range of base modifications. Each of these patents is
herein incorporated by reference in its entirety, and specifically for
their description of base modifications, their synthesis, their use, and
their incorporation into oligonucleotides and nucleic acids.

[0215]Nucleotide analogs can also include modifications of the sugar
moiety. Modifications to the sugar moiety would include natural
modifications of the ribose and deoxyribose as well as synthetic
modifications. Sugar modifications include but are not limited to the
following modifications at the 2' position: OH; F; O-, S-, or N-alkyl;
O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to
C10, alkyl or C2 to C10 alkenyl and alkynyl. 2' sugar modifications also
include but are not limited to --O[(CH2)nO]mCH3,
--O(CH2)nOCH3, --O(CH2)nNH2, --O(CH2)nCH3,
--O(CH2)n--ONH2, and
--O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to
about 10.

[0216]Other modifications at the 2' position include but are not limited
to: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3,
OCF3, SOCH3, SO2CH3, ONO2, NO2, N3,
NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a reporter
group, an intercalator, a group for improving the pharmacokinetic
properties of an oligonucleotide, or a group for improving the
pharmacodynamic properties of an oligonucleotide, and other substituents
having similar properties. Similar modifications can also be made at
other positions on the sugar, particularly the 3' position of the sugar
on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Modified sugars would also include
those that contain modifications at the bridging ring oxygen, such as
CH2 and S, Nucleotide sugar analogs can also have sugar mimetics
such as cyclobutyl moieties in place of the pentofuranosyl sugar. There
are numerous United States patents that teach the preparation of such
modified sugar structures such as U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920,
each of which is herein incorporated by reference in its entirety, and
specifically for their description of modified sugar structures, their
synthesis, their use, and their incorporation into nucleotides,
oligonucleotides and nucleic acids.

[0217]Nucleotide analogs can also be modified at the phosphate moiety.
Modified phosphate moieties include but are not limited to those that can
be modified so that the linkage between two nucleotides contains a
phosphorothioate, chiral phosphorothioate, phosphorodithioate,
phosphotriester, aminoalkylphosphotriester, methyl and other alkyl
phosphonates including 3'-alkylene phosphonate and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates. It is understood that these phosphate or modified
phosphate linkages between two nucleotides can be through a 3'-5' linkage
or a 2'-5' linkage, and the linkage can contain inverted polarity such as
3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free
acid forms are also included. Numerous United States patents teach how to
make and use nucleotides containing modified phosphates and include but
are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein
incorporated by reference its entirety, and specifically for their
description of modified phosphates, their synthesis, their use, and their
incorporation into nucleotides, oligonucleotides and nucleic acids.

[0218]It is understood that nucleotide analogs need only contain a single
modification, but can also contain multiple modifications within one of
the moieties or between different moieties.

[0219]Nucleotide substitutes are molecules having similar functional
properties to nucleotides, but which do not contain a phosphate moiety,
such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules
that will recognize and hybridize to (base pair to) complementary nucleic
acids in a Watson-Crick or Hoogsteen manner, but which are linked
together through a moiety other than a phosphate moiety. Nucleotide
substitutes are able to conform to a double helix type structure when
interacting with the appropriate target nucleic acid.

[0220]Nucleotide substitutes are nucleotides or nucleotide analogs that
have had the phosphate moiety and/or sugar moieties replaced. Nucleotide
substitutes do not contain a standard phosphorus atom. Substitutes for
the phosphate can be for example, short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic or
heterocyclic internucleoside linkages. These include those having
morpholino linkages (formed in part from the sugar portion of a
nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone
backbones; formacetyl and thioformacetyl backbones; methylene formacetyl
and thioformacetyl backbones; alkene containing backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N, O, S
and CH2 component parts. Numerous United States patents disclose how
to make and use these types of phosphate replacements and include but are
not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;
5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;
5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;
5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;
5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which
is herein incorporated by reference its entirety, and specifically for
their description of phosphate replacements, their synthesis, their use,
and their incorporation into nucleotides, oligonucleotides and nucleic
acids.

[0221]It is also understood in a nucleotide substitute that both the sugar
and the phosphate moieties of the nucleotide can be replaced, by for
example an amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.
5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA
molecules, each of which is herein incorporated by reference. (See also
Nielsen et al., Science 254:1497-1500 (1991)).

[0222]Oligonucleotides and nucleic acids can be comprised of nucleotides
and can be made up of different types of nucleotides or the same type of
nucleotides. For example, one or more of the nucleotides in an
oligonucleotide can be ribonucleotides, 2'-O-methyl ribonucleotides, or a
mixture of ribonucleotides and 2'-O-methyl ribonucleotides; about 10% to
about 50% of the nucleotides can be ribonucleotides, 2'-O-methyl
ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl
ribonucleotides; about 50% or more of the nucleotides can be
ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of
ribonucleotides and 2'-O-methyl ribonucleotides; or all of the
nucleotides are ribonucleotides, 2'-O-methyl ribonucleotides, or a
mixture of ribonucleotides and 2'-β-methyl ribonucleotides. Such
oligonucleotides and nucleic acids can be referred to as chimeric
oligonucleotides and chimeric nucleic acids.

L. Solid Supports

[0223]Solid supports are solid-state substrates or supports with which
molecules (such as trigger molecules) and riboswitches (or other
components used in, or produced by, the disclosed methods) can be
associated. Riboswitches and other molecules can be associated with solid
supports directly or indirectly. For example, analytes (e.g., trigger
molecules, test compounds) can be bound to the surface of a solid support
or associated with capture agents (e.g., compounds or molecules that bind
an analyte) immobilized on solid supports. As another example,
riboswitches can be bound to the surface of a solid support or associated
with probes immobilized on solid supports. An array is a solid support to
which multiple riboswitches, probes or other molecules have been
associated in an array, grid, or other organized pattern.

[0224]Solid-state substrates for use in solid supports can include any
solid material with which components can be associated, directly or
indirectly. This includes materials such as acrylamide, agarose,
cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl
acetate, polypropylene, polymethacrylate, polyethylene, polyethylene
oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon,
silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, functionalized silane, polypropylfumerate, collagen,
glycosaminoglycans, and polyamino acids. Solid-state substrates can have
any useful form including thin film, membrane, bottles, dishes, fibers,
woven fibers, shaped polymers, particles, beads, microparticles, or a
combination. Solid-state substrates and solid supports can be porous or
non-porous. A chip is a rectangular or square small piece of material.
Preferred forms for solid-state substrates are thin films, beads, or
chips. A useful form for a solid-state substrate is a microtiter dish. In
some embodiments, a multiwell glass slide can be employed.

[0225]An array can include a plurality of riboswitches, trigger molecules,
other molecules, compounds or probes immobilized at identified or
predefined locations on the solid support. Each predefined location on
the solid support generally has one type of component (that is, all the
components at that location are the same). Alternatively, multiple types
of components can be immobilized in the same predefined location on a
solid support. Each location will have multiple copies of the given
components. The spatial separation of different components on the solid
support allows separate detection and identification.

[0226]Although useful, it is not required that the solid support be a
single unit or structure. A set of riboswitches, trigger molecules, other
molecules, compounds and/or probes can be distributed over any number of
solid supports. For example, at one extreme, each component can be
immobilized in a separate reaction tube or container, or on separate
beads or microparticles.

[0227]Methods for immobilization of oligonucleotides to solid-state
substrates are well established. Oligonucleotides, including address
probes and detection probes, can be coupled to substrates using
established coupling methods. For example, suitable attachment methods
are described by Pease et al., Proc. Natl. Acad. Sci. USA 91(10:5022-5026
(1994), and Khrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A
method for immobilization of 3'-amine oligonucleotides on casein-coated
slides is described by Stimpson et al., Proc. Natl. Acad. Sci. USA
92:6379-6383 (1995). A useful method of attaching oligonucleotides to
solid-state substrates is described by Guo et al., Nucleic Acids Res.
22:5456-5465 (1994).

[0228]Each of the components (for example, riboswitches, trigger
molecules, or other molecules) immobilized on the solid support can be
located in a different predefined region of the solid support. The
different locations can be different reaction chambers. Each of the
different predefined regions can be physically separated from each other
of the different regions. The distance between the different predefined
regions of the solid support can be either fixed or variable. For
example, in an array, each of the components can be arranged at fixed
distances from each other, while components associated with beads will
not be in a fixed spatial relationship. In particular, the use of
multiple solid support units (for example, multiple beads) will result in
variable distances.

[0229]Components can be associated or immobilized on a solid support at
any density. Components can be immobilized to the solid support at a
density exceeding 400 different components per cubic centimeter. Arrays
of components can have any number of components. For example, an array
can have at least 1,000 different components immobilized on the solid
support, at least 10,000 different components immobilized on the solid
support, at least 100,000 different components immobilized on the solid
support, or at least 1,000,000 different components immobilized on the
solid support.

M. Kits

[0230]The materials described above as well as other materials can be
packaged together in any suitable combination as a kit useful for
performing, or aiding in the performance of, the disclosed method. It is
useful if the kit components in a given kit are designed and adapted for
use together in the disclosed method. For example disclosed are kits for
detecting compounds, the kit comprising one or more biosensor
riboswitches. The kits also can contain reagents and labels for detecting
activation of the riboswitches.

N. Mixtures

[0231]Disclosed are mixtures formed by performing or preparing to perform
the disclosed method. For example, disclosed are mixtures comprising
riboswitches and trigger molecules.

[0232]Whenever the method involves mixing or bringing into contact
compositions or components or reagents, performing the method creates a
number of different mixtures. For example, if the method includes 3
mixing steps, after each one of these steps a unique mixture is formed if
the steps are performed separately. In addition, a mixture is formed at
the completion of all of the steps regardless of how the steps were
performed. The present disclosure contemplates these mixtures, obtained
by the performance of the disclosed methods as well as mixtures
containing any disclosed reagent, composition, or component, for example,
disclosed herein.

O. Systems

[0233]Disclosed are systems useful for performing, or aiding in the
performance of, the disclosed method. Systems generally comprise
combinations of articles of manufacture such as structures, machines,
devices, and the like, and compositions, compounds, materials, and the
like. Such combinations that are disclosed or that are apparent from the
disclosure are contemplated. For example, disclosed and contemplated are
systems comprising biosensor riboswitches, a solid support and a
signal-reading device.

P. Data Structures and Computer Control

[0234]Disclosed are data structures used in, generated by, or generated
from, the disclosed method. Data structures generally are any form of
data, information, and/or objects collected, organized, stored, and/or
embodied in a composition or medium. Riboswitch structures and activation
measurements stored in electronic form, such as in RAM or on a storage
disk, is a type of data structure.

[0235]The disclosed method, or any part thereof or preparation therefor,
can be controlled, managed, or otherwise assisted by computer control.
Such computer control can be accomplished by a computer controlled
process or method, can use and/or generate data structures, and can use a
computer program. Such computer control, computer controlled processes,
data structures, and computer programs are contemplated and should be
understood to be disclosed herein.

Methods

[0236]Disclosed are methods for activating, deactivating or blocking a
riboswitch. Such methods can involve, for example, bringing into contact
a riboswitch and a compound or trigger molecule that can activate,
deactivate or block the riboswitch. Riboswitches function to control gene
expression through the binding or removal of a trigger molecule.
Compounds can be used to activate, deactivate or block a riboswitch. The
trigger molecule for a riboswitch (as well as other activating compounds)
can be used to activate a riboswitch. Compounds other than the trigger
molecule generally can be used to deactivate or block a riboswitch.
Riboswitches can also be deactivated by, for example, removing trigger
molecules from the presence of the riboswitch. Thus, the disclosed method
of deactivating a riboswitch can involve, for example, removing a trigger
molecule (or other activating compound) from the presence or contact with
the riboswitch. A riboswitch can be blocked by, for example, binding of
an analog of the trigger molecule that does not activate the riboswitch.

[0237]Also disclosed are methods for altering expression of an RNA
molecule, or of a gene encoding an RNA molecule, where the RNA molecule
includes a riboswitch, by bringing a compound into contact with the RNA
molecule. Riboswitches function to control gene expression through the
binding or removal of a trigger molecule. Thus, subjecting an RNA
molecule of interest that includes a riboswitch to conditions that
activate, deactivate or block the riboswitch can be used to alter
expression of the RNA. Expression can be altered as a result of, for
example, termination of transcription or blocking of ribosome binding to
the RNA. Binding of a trigger molecule can, depending on the nature of
the riboswitch, reduce or prevent expression of the RNA molecule or
promote or increase expression of the RNA molecule.

[0238]Also disclosed are methods for regulating expression of a naturally
occurring gene or RNA that contains a riboswitch by activating,
deactivating or blocking the riboswitch. If the gene is essential for
survival of a cell or organism that harbors it, activating, deactivating
or blocking the riboswitch can result in death, stasis or debilitation of
the cell or organism. For example, activating a naturally occurring
riboswitch in a naturally occurring gene that is essential to survival of
a microorganism can result in death of the microorganism (if activation
of the riboswitch turns off or represses expression). This is one basis
for the use of the disclosed compounds and methods for antimicrobial and
antibiotic effects. The compounds that have these antimicrobial effects
are considered to be bacteriostatic or bacteriocidal.

[0239]Also disclosed are methods for selecting and identifying compounds
that can activate, deactivate or block a riboswitch. Activation of a
riboswitch refers to the change in state of the riboswitch upon binding
of a trigger molecule. A riboswitch can be activated by compounds other
than the trigger molecule and in ways other than binding of a trigger
molecule. The term trigger molecule is used herein to refer to molecules
and compounds that can activate a riboswitch. This includes the natural
or normal trigger molecule for the riboswitch and other compounds that
can activate the riboswitch. Natural or normal trigger molecules are the
trigger molecule for a given riboswitch in nature or, in the case of some
non-natural riboswitches, the trigger molecule for which the riboswitch
was designed or with which the riboswitch was selected (as in, for
example, in vitro selection or in vitro evolution techniques).
Non-natural trigger molecules can be referred to as non-natural trigger
molecules.

[0240]Also disclosed are methods of killing or inhibiting bacteria,
comprising contacting the bacteria with a compound disclosed herein or
identified by the methods disclosed herein.

[0241]Also disclosed are methods of identifying compounds that activate,
deactivate or block a riboswitch. For example, compounds that activate a
riboswitch can be identified by bringing into contact a test compound and
a riboswitch and assessing activation of the riboswitch. If the
riboswitch is activated, the test compound is identified as a compound
that activates the riboswitch. Activation of a riboswitch can be assessed
in any suitable manner. For example, the riboswitch can be linked to a
reporter RNA and expression, expression level, or change in expression
level of the reporter RNA can be measured in the presence and absence of
the test compound. As another example, the riboswitch can include a
conformation dependent label, the signal from which changes depending on
the activation state of the riboswitch. Such a riboswitch preferably uses
an aptamer domain from or derived from a naturally occurring riboswitch.
As can be seen, assessment of activation of a riboswitch can be performed
with the use of a control assay or measurement or without the use of a
control assay or measurement. Methods for identifying compounds that
deactivate a riboswitch can be performed in analogous ways.

[0242]In addition to the methods disclosed elsewhere herein,
identification of compounds that block a riboswitch can be accomplished
in any suitable manner. For example, an assay can be performed for
assessing activation or deactivation of a riboswitch in the presence of a
compound known to activate or deactivate the riboswitch and in the
presence of a test compound. If activation or deactivation is not
observed as would be observed in the absence of the test compound, then
the test compound is identified as a compound that blocks activation or
deactivation of the riboswitch.

[0243]Also disclosed are methods of detecting compounds using biosensor
riboswitches. The method can include bringing into contact a test sample
and a biosensor riboswitch and assessing the activation of the biosensor
riboswitch. Activation of the biosensor riboswitch indicates the presence
of the trigger molecule for the biosensor riboswitch in the test sample.
Biosensor riboswitches are engineered riboswitches that produce a
detectable signal in the presence of their cognate trigger molecule.
Useful biosensor riboswitches can be triggered at or above threshold
levels of the trigger molecules. Biosensor riboswitches can be designed
for use in vivo or in vitro. For example, lysine biosensor riboswitches
operably linked to a reporter RNA that encodes a protein that serves as
or is involved in producing a signal can be used in vivo by engineering a
cell or organism to harbor a nucleic acid construct encoding the
riboswitch/reporter RNA. An example of a biosensor riboswitch for use in
vitro is a lysine riboswitch that includes a conformation dependent
label, the signal from which changes depending on the activation state of
the riboswitch. Such a biosensor riboswitch preferably uses an aptamer
domain from or derived from a naturally occurring lysine riboswitch.

[0244]Also disclosed are compounds made by identifying a compound that
activates, deactivates or blocks a riboswitch and manufacturing the
identified compound. This can be accomplished by, for example, combining
compound identification methods as disclosed elsewhere herein with
methods for manufacturing the identified compounds. For example,
compounds can be made by bringing into contact a test compound and a
riboswitch, assessing activation of the riboswitch, and, if the
riboswitch is activated by the test compound, manufacturing the test
compound that activates the riboswitch as the compound.

[0245]Also disclosed are compounds made by checking activation,
deactivation or blocking of a riboswitch by a compound and manufacturing
the checked compound. This can be accomplished by, for example, combining
compound activation, deactivation or blocking assessment methods as
disclosed elsewhere herein with methods for manufacturing the checked
compounds. For example, compounds can be made by bringing into contact a
test compound and a riboswitch, assessing activation of the riboswitch,
and, if the riboswitch is activated by the test compound, manufacturing
the test compound that activates the riboswitch as the compound. Checking
compounds for their ability to activate, deactivate or block a riboswitch
refers to both identification of compounds previously unknown to
activate, deactivate or block a riboswitch and to assessing the ability
of a compound to activate, deactivate or block a riboswitch where the
compound was already known to activate, deactivate or block the
riboswitch.

[0246]Disclosed is a method of detecting a compound of interest, the
method comprising bringing into contact a sample and a lysine riboswitch,
wherein the riboswitch is activated by the compound of interest, wherein
the riboswitch produces a signal when activated by the compound of
interest, wherein the riboswitch produces a signal when the sample
contains the compound of interest. The riboswitch can change conformation
when activated by the compound of interest, wherein the change in
conformation produces a signal via a conformation dependent label. The
riboswitch can change conformation when activated by the compound of
interest, wherein the change in conformation causes a change in
expression of an RNA linked to the riboswitch, wherein the change in
expression produces a signal. The signal can be produced by a reporter
protein expressed from the RNA linked to the riboswitch.

[0247]Disclosed is a method comprising (a) testing a compound for
inhibition of gene expression of a gene encoding an RNA comprising a
riboswitch, wherein the inhibition is via the riboswitch, and (b)
inhibiting gene expression by bringing into contact a cell and a compound
that inhibited gene expression in step (a), wherein the cell comprises a
gene encoding an RNA comprising a riboswitch, wherein the compound
inhibits expression of the gene by binding to the riboswitch.

A. Identification of Antimicrobial Compounds

[0248]Riboswitches are a new class of structured RNAs that have evolved
for the purpose of binding small organic molecules. The natural binding
pocket of riboswitches can be targeted with metabolite analogs or by
compounds that mimic the shape-space of the natural metabolite. The small
molecule ligands of riboswitches provide useful sites for derivitization
to produce drug candidates. Distribution of some riboswitches is shown in
Table 1 of U.S. Application Publication No. 2005-0053951. Once a class of
riboswitch has been identified and its potential as a drug target
assessed, such as the lysine riboswitch, candidate molecules can be
identified.

[0249]The emergence of drug-resistant stains of bacteria highlights the
need for the identification of new classes of antibiotics.
Anti-riboswitch drugs represent a mode of anti-bacterial action that is
of considerable interest for the following reasons. Riboswitches control
the expression of genes that are critical for fundamental metabolic
processes. Therefore manipulation of these gene control elements with
drugs yields new antibiotics. These antimicrobial agents can be
considered to be bacteriostatic, or bacteriocidal. Riboswitches also
carry RNA structures that have evolved to selectively bind metabolites,
and therefore these RNA receptors make good drug targets as do protein
enzymes and receptors. Furthermore, it has been shown that two
antimicrobial compounds (discussed above) kill bacteria by deactivating
the antibiotics resistance to emerge through mutation of the RNA target.

[0250]A compound can be identified as activating a riboswitch or can be
determined to have riboswitch activating activity if the signal in a
riboswitch assay is increased in the presence of the compound by at least
1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 50%, 75%, 100%, 125%, 150%, 175%,
200%, 250%, 300%, 400%, or 500% compared to the same riboswitch assay in
the absence of the compound (that is, compared to a control assay). The
riboswitch assay can be performed using any suitable riboswitch
construct. Riboswitch constructs that are particularly useful for
riboswitch activation assays are described elsewhere herein. The
identification of a compound as activating a riboswitch or as having a
riboswitch activation activity can be made in terms of one or more
particular riboswitches, riboswitch constructs or classes of
riboswitches. For convenience, compounds identified as activating a
lysine riboswitch or having riboswitch activating activity for a lysine
riboswitch can be so identified for particular lysine riboswitches, such
as the lysine riboswitches found in Bacillus anthracis or B. subtilis.

B. Methods of Using Antimicrobial Compounds

[0251]Disclosed herein are in vivo and in vitro anti-bacterial methods. By
"anti-bacterial" is meant inhibiting or preventing bacterial growth,
killing bacteria, or reducing the number of bacteria. Thus, disclosed is
a method of inhibiting or preventing bacterial growth comprising
contacting a bacterium with an effective amount of one or more compounds
disclosed herein. Additional structures for the disclosed compounds are
provided herein.

[0252]Disclosed herein is also a method of inhibiting growth of a cell,
such as a bacterial cell, that is in a subject, the method comprising
administering an effective amount of a compound as disclosed herein to
the subject. This can result in the compound being brought into contact
with the cell. The subject can have, for example, a bacterial infection,
and the bacterial cells can be inhibited by the compound. The bacteria
can be any bacteria, such as bacteria from the genus Bacillus,
Acinetobacter, Actinobacillus, Clostridium, Desulfitobacterium,
Enterococcus, Erwinia, Escherichia, Exiguobacterium, Fusobacterium,
Geobacillus, Haemophilus, Klebsiella, Idiomarina, Lactobacillus,
Lactococcus, Leuconostoc, Listeria, Moorella, Mycobacterium,
Oceanobacillus, Oenococcus, Pasteurella, Pediococcus, Pseudomonas,
Shewanella, Shigella, Solibacter, Staphylococcus, Streptococcus,
Thermoanaerobacter, Thermotoga, and Vibrio, for example. The bacteria can
be, for example, Actinobacillus pleuropneumoniae, Bacillus anthracis,
Bacillus cereus, Bacillus clausii, Bacillus halodurans, Bacillus
licheniformis, Bacillus subtilis, Bacillus thuringiensis, Clostridium
acetobutylicum, Clostridium difficile, Clostridium perfringens,
Clostridium tetani, Clostridium thermocellum, Desulfitobacterium
hafniense, Enterococcus faecalis, Erwinia carotovora, Escherichia coli,
Exiguobacterium sp., Fusobacterium nucleatum, Geobacillus kaustophilus,
Haemophilus ducreyi, Haemophilus influenzae, Haemophilus somnus,
Idiomarina loihiensis, Lactobacillus acidophilus, Lactobacillus casei,
Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus
johnsonii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc
mesenteroides, Listeria innocua, Listeria monocytogenes, Moorella
thermoacetica, Oceanobacillus iheyensis, Oenococcus oeni, Pasteurella
multocida, Pediococcus pentosaceus, Shewanella oneidensis, Shigella
flexneri, Solibacter usitatus, Staphylococcus aureus, Staphylococcus
epidermidis, Thermoanaerobacter tengcongensis, Thermotoga maritima,
Vibrio cholerae, Vibrio fischeri, Vibrio parahaemolyticus, or Vibrio
vulnificus. Bacterial growth can also be inhibited in any context in
which bacteria are found. For example, bacterial growth in fluids,
biofilms, and on surfaces can be inhibited. The compounds disclosed
herein can be administered or used in combination with any other compound
or composition. For example, the disclosed compounds can be administered
or used in combination with another antimicrobial compound.

[0253]"Inhibiting bacterial growth" is defined as reducing the ability of
a single bacterium to divide into daughter cells, or reducing the ability
of a population of bacteria to form daughter cells. The ability of the
bacteria to reproduce can be reduced by about 10%, about 20%, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100%
or more.

[0254]Also provided is a method of inhibiting the growth of and/or killing
a bacterium or population of bacteria comprising contacting the bacterium
with one or more of the compounds disclosed and described herein.

[0255]"Killing a bacterium" is defined as causing the death of a single
bacterium, or reducing the number of a plurality of bacteria, such as
those in a colony. When the bacteria are referred to in the plural form,
the "killing of bacteria" is defined as cell death of a given population
of bacteria at the rate of 10% of the population, 20% of the population,
30% of the population, 40% of the population, 50% of the population, 60%
of the population, 70% of the population, 80% of the population, 90% of
the population, or less than or equal to 100% of the population.

[0256]The compounds and compositions disclosed herein have anti-bacterial
activity in vitro or in vivo, and can be used in conjunction with other
compounds or compositions, which can be bactericidal as well.

[0257]By the term "therapeutically effective amount" of a compound as
provided herein is meant a nontoxic but sufficient amount of the compound
to provide the desired reduction in one or more symptoms. As will be
pointed out below, the exact amount of the compound required will vary
from subject to subject, depending on the species, age, and general
condition of the subject, the severity of the disease that is being
treated, the particular compound used, its mode of administration, and
the like. Thus, it is not possible to specify an exact "effective
amount." However, an appropriate effective amount may be determined by
one of ordinary skill in the art using only routine experimentation.

[0258]The compositions and compounds disclosed herein can be administered
in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically
acceptable" is meant a material that is not biologically or otherwise
undesirable, i.e., the material may be administered to a subject without
causing any undesirable biological effects or interacting in a
deleterious manner with any of the other components of the pharmaceutical
composition in which it is contained. The carrier would naturally be
selected to minimize any degradation of the active ingredient and to
minimize any adverse side effects in the subject, as would be well known
to one of skill in the art.

[0259]The compositions or compounds disclosed herein can be administered
orally, parenterally (e.g., intravenously), by intramuscular injection,
by intraperitoneal injection, transdermally, extracorporeally, topically
or the like, including topical intranasal administration or
administration by inhalant. As used herein, "topical intranasal
administration" means delivery of the compositions into the nose and
nasal passages through one or both of the nares and can comprise delivery
by a spraying mechanism or droplet mechanism, or through aerosolization
of the nucleic acid or vector. Administration of the compositions by
inhalant can be through the nose or mouth via delivery by a spraying or
droplet mechanism. Delivery can also be directly to any area of the
respiratory system (e.g., lungs) via intubation. The exact amount of the
compositions required will vary from subject to subject, depending on the
species, age, weight and general condition of the subject, the severity
of the allergic disorder being treated, the particular nucleic acid or
vector used, its mode of administration and the like. Thus, it is not
possible to specify an exact amount for every composition. However, an
appropriate amount can be determined by one of ordinary skill in the art
using only routine experimentation given the teachings herein.

[0260]Parenteral administration of the composition or compounds, if used,
is generally characterized by injection. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions, solid
forms suitable for solution of suspension in liquid prior to injection,
or as emulsions. A more recently revised approach for parenteral
administration involves use of a slow release or sustained release system
such that a constant dosage is maintained. See, e.g., U.S. Pat. No.
3,610,795, which is incorporated by reference herein.

[0261]The compositions and compounds disclosed herein can be used
therapeutically in combination with a pharmaceutically acceptable
carrier. Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R.
Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an
appropriate amount of a pharmaceutically-acceptable salt is used in the
formulation to render the formulation isotonic. Examples of the
pharmaceutically-acceptable carrier include, but are not limited to,
saline, Ringer's solution and dextrose solution. The pH of the solution
is preferably from about 5 to about 8, and more preferably from about 7
to about 7.5. Further carriers include sustained release preparations
such as semipermeable matrices of solid hydrophobic polymers containing
the antibody, which matrices are in the form of shaped articles, e.g.,
films, liposomes or microparticles. It will be apparent to those persons
skilled in the art that certain carriers may be more preferable depending
upon, for instance, the route of administration and concentration of
composition being administered.

[0262]Pharmaceutical carriers are known to those skilled in the art. These
most typically would be standard carriers for administration of drugs to
humans, including solutions such as sterile water, saline, and buffered
solutions at physiological pH. The compositions can be administered
intramuscularly or subcutaneously. Other compounds will be administered
according to standard procedures used by those skilled in the art.

[0263]Pharmaceutical compositions may include carriers, thickeners,
diluents, buffers, preservatives, surface active agents and the like in
addition to the molecule of choice. Pharmaceutical compositions may also
include one or more active ingredients such as antimicrobial agents,
antiinflammatory agents, anesthetics, and the like.

[0264]The pharmaceutical composition may be administered in a number of
ways depending on whether local or systemic treatment is desired, and on
the area to be treated. Administration may be topically (including
ophthalmically, vaginally, rectally, intranasally), orally, by
inhalation, or parenterally, for example by intravenous drip,
subcutaneous, intraperitoneal or intramuscular injection. The disclosed
antibodies can be administered intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity, or transdermally.

[0265]Preparations for parenteral administration include sterile aqueous
or non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable
oils such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose, dextrose
and sodium chloride, lactated Ringer's, or fixed oils. Intravenous
vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for
example, antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.

[0266]Formulations for topical administration may include ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily bases,
thickeners and the like may be necessary or desirable.

[0267]Compositions for oral administration include powders or granules,
suspensions or solutions in water or non-aqueous media, capsules,
sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,
dispersing aids or binders may be desirable.

[0269]Therapeutic compositions as disclosed herein may also be delivered
by the use of monoclonal antibodies as individual carriers to which the
compound molecules are coupled. The therapeutic compositions of the
present disclosure may also be coupled with soluble polymers as
targetable drug carriers. Such polymers can include, but are not limited
to, polyvinyl-pyrrolidone, pyran copolymer,
polyhydroxypropylmethacryl-amidephenol,
polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysine
substituted with palmitoyl residues. Furthermore, the therapeutic
compositions of the present disclosure may be coupled to a class of
biodegradable polymers useful in achieving controlled release of a drug,
for example, polylactic acid, polyepsilon caprolactone, polyhydroxy
butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,
polycyanoacrylates and cross-linked or amphipathic block copolymers of
hydrogels.

[0270]Preferably at least about 3%, more preferably about 10%, more
preferably about 20%, more preferably about 30%, more preferably about
50%, more preferably 75% and even more preferably about 100% of the
bacterial infection is reduced due to the administration of the compound.
A reduction in the infection is determined by such parameters as reduced
white blood cell count, reduced fever, reduced inflammation, reduced
number of bacteria, or reduction in other indicators of bacterial
infection. To increase the percentage of bacterial infection reduction,
the dosage can increase to the most effective level that remains
non-toxic to the subject.

[0271]As used throughout, "subject" refers to an individual. Preferably,
the subject is a mammal such as a non-human mammal or a primate, and,
more preferably, a human. "Subjects" can include domesticated animals
(such as cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep,
goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig,
etc.) and fish.

[0272]A "bacterial infection" is defined as the presence of bacteria in a
subject or sample. Such bacteria can be an outgrowth of naturally
occurring bacteria in or on the subject or sample, or can be due to the
invasion of a foreign organism.

[0273]The compounds disclosed herein can be used in the same manner as
antibiotics. Uses of antibiotics are well established in the art. One
example of their use includes treatment of animals. When needed, the
disclosed compounds can be administered to the animal via injection or
through feed or water, usually with the professional guidance of a
veterinarian or nutritionist. They are delivered to animals either
individually or in groups, depending on the circumstances such as disease
severity and animal species. Treatment and care of the entire herd or
flock may be necessary if all animals are of similar immune status and
all are exposed to the same disease-causing microorganism.

[0274]Another example of a use for the compounds includes reducing a
microbial infection of an aquatic animal, comprising the steps of
selecting an aquatic animal having a microbial infection, providing an
antimicrobial solution comprising a compound as disclosed, chelating
agents such as EDTA, TRIENE, adding a pH buffering agent to the solution
and adjusting the pH thereof to a value of between about 7.0 and about
9.0, immersing the aquatic animal in the solution and leaving the aquatic
animal therein for a period that is effective to reduce the microbial
burden of the animal, removing the aquatic animal from the solution and
returning the animal to water not containing the solution. The immersion
of the aquatic animal in the solution containing the EDTA, a compound as
disclosed, and TRIENE and pH buffering agent may be repeated until the
microbial burden of the animal is eliminated. (U.S. Pat. No. 6,518,252).

[0275]Other uses of the compounds disclosed herein include, but are not
limited to, dental treatments and purification of water (this can include
municipal water, sewage treatment systems, potable and non-potable water
supplies, and hatcheries, for example).

Specific Embodiments

[0276]Disclosed herein is a method of inhibiting gene expression, the
method comprising (a) bringing into contact a compound and a cell, (b)
wherein the compound has the structure of Formula I:

##STR00004##

[0277]wherein R2 and R3 are each independently positively
charged, can serve as a hydrogen bond donor, or both,

[0278]wherein R1 is negatively charged, R4 is negatively
charged, or R1 and R4 are in a resonance hybrid with a net
negative charge,

[0285]wherein can each independently represent a single or double bond,
and

[0286]wherein the compound is not lysine, and wherein the cell comprises a
gene encoding an RNA comprising a lysine-responsive riboswitch, wherein
the compound inhibits expression of the gene by binding to the
lysine-responsive riboswitch.

[0287]R3 can be positively charged and can serve as a hydrogen bond
donor. R5 can be uncharged. R9 can be C, O, or S. The pKa
of R3 can be 7 or higher. R13 can be positively charged, and
can serve as a hydrogen bond donor, or both.

[0288]In one example, R6, R7, R8, R9, R10 and
R11 are not all simultaneously C, CH, or CH2.

[0289]In another example, R1, R2, R3, R4 and R9
are not simultaneously O, NH3.sup.+, NH3.sup.+, O and S,
respectively. Furthermore, in another example, R1, R2, R3,
and R4 are not simultaneously O, H, NH3.sup.+, and O,
respectively. In another example, R1, R2, R3, R4 and
R9 are not simultaneously CO2-, NH3.sup.+,
NH3.sup.+, and H, respectively. In a further example, R1,
R2, R3, R4 and R11 are not simultaneously O,
NH3.sup.+, NH3.sup.+, O and C--CO2-, respectively. In
a further example, R1, R2, R3, and R4 are not
simultaneously NHOH, NH3.sup.+, NH3.sup.+, O and S,
respectively.

[0293]In a further example, R10 can be N, NH, O, or S. In a further
example, R7 can be CH.

[0294]The cell can be identified as being in need of inhibited gene
expression. The cell can be a bacterial cell, for example, and the
compound can kill or inhibit the growth of the bacterial cell. The
compound and the cell can be brought into contact by administering the
compound to a subject. In one example, the compound is not a substrate
for enzymes of the subject that have lysine as a substrate. The compound
can also not be a substrate for enzymes of the subject that alter lysine.
The compound can also not be a substrate for enzymes of the subject that
metabolize lysine. The compound can also not be a substrate for enzymes
of the subject that catabolize lysine. The cell can be a bacterial cell
in the subject, wherein the compound kills or inhibits the growth of the
bacterial cell.

[0295]Disclosed herein is a compound having the structure of Formula I:

##STR00005##

[0296]wherein R2 and R3 are each independently positively
charged, can serve as a hydrogen bond donor, or both,

[0297]wherein R1 is negatively charged, R4 is negatively
charged, or R1 and R4 are in a resonance hybrid with a net
negative charge,

[0304]wherein can each independently represent a single or double bond,
and

[0305]wherein the compound is not lysine.

[0306]R3 can be positively charged and can serve as a hydrogen bond
donor. R5 can be uncharged. R9 can be C, O, or S. The pKa
of R3 can be 7 or higher. R13 can be positively charged, and
can serve as a hydrogen bond donor, or both.

[0307]In one example, R6, R7, R8, R9, R10 and
R11 are not all simultaneously C, CH, or CH2.

[0308]In another example, R1, R2, R3, R4 and R9
are not simultaneously O, NH3.sup.+, NH3.sup.+, O and S,
respectively. Furthermore, in another example, R1, R2, R3,
and R4 are not simultaneously O, H, NH3.sup.+, and O,
respectively. In another example, R1, R2, R3, R4 and
R9 are not simultaneously CO2-, NH3.sup.+,
NH3.sup.+, and H, respectively. In a further example, R1,
R2, R3, R4 and R11 are not simultaneously O,
NH3.sup.+, NH3.sup.+, O and C--CO2-, respectively. In
a further example, R1, R2, R3, and R4 are not
simultaneously NHOH, NH3.sup.+, NH3.sup.+, O and S,
respectively.

[0312]In a further example, R10 can be N, NH, O, or S. In a further
example, R7 can be CH.

[0313]Further disclosed is a composition comprising the compound described
above and a regulatable gene expression construct comprising a nucleic
acid molecule encoding an RNA comprising a lysine riboswitch operably
linked to a coding region, wherein the lysine riboswitch regulates
expression of the RNA, wherein the lysine riboswitch and coding region
are heterologous. The lysine riboswitch can produce a signal when
activated by the compound. For example, the riboswitch can change
conformation when activated by the compound, and the change in
conformation can produce a signal via a conformation dependent label.
Furthermore, the riboswitch can change conformation when activated by the
compound, wherein the change in conformation causes a change in
expression of the coding region linked to the riboswitch, wherein the
change in expression produces a signal. The signal can be produced by a
reporter protein expressed from the coding region linked to the
riboswitch.

[0314]Also disclosed is a method comprising: (a) testing the compound as
described above for inhibition of gene expression of a gene encoding an
RNA comprising a lysine riboswitch, wherein the inhibition is via the
lysine riboswitch, and (b) inhibiting gene expression by bringing into
contact a cell and a compound that inhibited gene expression in step (a),
wherein the cell comprises a gene encoding an RNA comprising the lysine
riboswitch, wherein the compound inhibits expression of the gene by
binding to the lysine riboswitch.

[0315]Further disclosed is a method of killing bacteria, comprising
contacting the bacteria with a compound disclosed above. Disclosed herein
is also a method of inhibiting growth of a cell, such as a bacterial
cell, that is in a subject, the method comprising administering an
effective amount of a compound as disclosed herein to the subject. This
can result in the compound being brought into contact with the cell. The
subject can have, for example, a bacterial infection, and the bacterial
cells can be the cells to be inhibited by the compound. The bacteria can
be any bacteria. Bacterial growth can also be inhibited in any context in
which bacteria are found. For example, bacterial growth in fluids,
biofilms, and on surfaces can be inhibited. The compounds disclosed
herein can be administered or used in combination with any other compound
or composition. For example, the disclosed compounds can be administered
or used in combination with another antimicrobial compound.

EXAMPLES

Example 1

Antibacterial Lysine Analogs that Target Lysine Riboswitches

[0316]Lysine riboswitches are bacterial RNA structures that sense the
concentration of lysine and regulate the expression of lysine
biosynthesis and transport genes. Members of this riboswitch class are
found in the 5'-untranslated region (5'-UTR) of messenger RNAs, where
they form highly selective receptors for lysine. Lysine binding to the
receptor stabilizes an mRNA tertiary structure that, in most cases,
causes transcription termination before the adjacent open reading frame
can be expressed. A lysine riboswitch can be used for antibacterial
therapy by designing compounds that bind the riboswitch and suppress
lysine biosynthesis and transport genes. As a test of this strategy,
several lysine analogs that bind to riboswitches and inhibit bacterial
growth have been identified. These results indicate that riboswitches can
serve as antibacterial drug targets.

[0317]In one example, a riboswitch-targeting compound is dissimilar to the
natural metabolite so that the drug can neither serve as a nutritional
supplement for the pathogen, nor interact with host enzymes that process
the natural metabolite. In order to identify functional groups where
modifications to lysine would be tolerated by lysine riboswitch
receptors, 12 lysine analogs were evaluated for their ability to bind the
riboswitch receptor from B. subtilis (FIG. 2a). The equilibrium
dissociation constant (KD) for each compound was established by
conducting in-line probing assays (Soukup 1999) with the 179-nucleotide
receptor domain (termed 179 lysC) of the riboswitch (FIG. 2b). In-line
probing reveals the ability of each internucleotide linkage to undergo
self-cleavage through an S.sub.N2P mechanism. As previously reported
(Sudarsan 2006) there are three regions of the receptor (A, B, and C,
FIG. 2b) where the extent of cleavage is reduced, compared to the pattern
in the absence of an added compound, indicating that the RNA undergoes a
structural change upon ligand binding. Quantitation of the fraction of
RNAs cleaved at each region as a function of ligand concentration gives a
reasonable measure of KD (FIG. 2c).

[0318]Five of the 12 analogs tested bind to 179 lysC with KD values
within 40 fold of the KD for lysine (360 nM), revealing that the
riboswitch can tolerate chemical modifications at certain positions of
its ligand. The observation that derivatives 1, 2, 4, and AEC (Sudarsan
2006) bind well indicates that additional modifications of various sizes
at the C4 position of lysine are likely to be tolerated by the receptor
and may even increase the affinity of the interaction. A large functional
group can also be accommodated on N6 (compounds 3 and 7), as long as this
amine still carries a hydrogen bond donor and a positive charge at
neutral pH. When N6 lacks a hydrogen bond donor (12) or has a pKa
less than 7 (6), no binding is observed. The poor affinity of 8 is
consistent with the previous observation that 5-hydroxylysine also fails
to bind to the riboswitch (Sudarsan 2006) showing that the RNA cannot
tolerate bulky C5 modifications to the ligand. Compounds 9 and 10 have
large modifications to the site chain atoms that add bulk and that are
expected to produce a pKa less than 7 at the amine equivalent to N6.
These differences can be sufficient to explain their poor affinities,
although other effects such as restricted conformation of the ligand can
also hinder binding. Finally, the riboswitch does not tolerate
modifications at N2 (5, 11), perhaps due to steric clash or to a change
in the ionic character of the nitrogen. Since removing N2 ablates binding
(Sudarsan 2006) either the charge or the hydrogen bonding character of N2
is needed for binding. The importance of the carboxylate, the
stereochemistry at C2, and the length of the amino acid side chain were
previously established (Sudarsan 2006). A summary of the molecular
recognition determinants for ligand binding to lysine riboswitch
receptors is depicted in FIG. 2d.

[0319]It was next determined whether any of the analogs inhibit the growth
of B. subtilis. At 100 μM in a chemically-defined minimal medium (see
Methods), five compounds slow bacterial growth (FIG. 3a,b). Of these,
only 1, 2, and 4 completely inhibit bacterial growth for 24 h (FIG. 3c).
Although 3 binds strongly to the lysine aptamer, this analog is the only
compound examined in this study other than lysine that supports the
growth of a lysine auxotroph strain (1A40) of B. subtilis (Supplementary
FIG. 3). Therefore, it appears that 3 is most likely serving as a
fortuitous precursor for lysine production, and this unexpected metabolic
conversion circumvents toxicity. Compound 7 also fails to inhibit cell
growth despite its strong binding to 179 lysC. 7 appears to be chemically
modified by the bacterium, or perhaps it cannot gain entry to the cell.
Either explanation is consistent with the observation that 7 does not
repress riboswitch-mediated reporter gene expression (FIG. 3c).

[0320]Compounds 8 and 9 are rejected by the lysine aptamer when tested in
vitro (FIG. 2a), and they do not repress expression of a reporter gene
controlled by the lysC riboswitch. However, both compounds exhibit a
modest level of growth inhibition activity (FIG. 3a,b). These findings
indicate that 8 and 9 inhibit bacterial growth by a mechanism that does
not involve the lysine riboswitch. Consistent with this is the
observation that 1 and 2 also inhibit the formation of viable spores
whereas 8 and 9 do not (Supplementary Table 2), showing that compounds
that trigger riboswitch function affect cellular processes that are
distinct from those that do not bind the riboswitch.

[0321]One likely explanation for the action of 1 and 2 is that they
inhibit growth and sporulation by binding to the lysine riboswitch and
repressing lysC, thereby depleting the bacteria of lysine and
2,3-dihydropicolinate. An alternate possibility is that they are
incorporated into proteins where they disrupt functional interactions.
Arguments have been presented for both possibilities as explanations for
why L-2-aminoethyl-cysteine (AEC) inhibits bacterial growth (Sudarsan
2006; Grundy 2003). Therefore, the mechanism of growth inhibition for 1,
2, and 4 were more carefully evaluated.

[0322]To determine if the lysine analogs repress gene expression, a
bacterial strain was constructed in which a copy of the lysC lysine
riboswitch was cloned upstream of a lacZ reporter gene and transformed
into the amyE locus of B. subtilis. As expected, β-galactosidase
expression is strongly repressed by lysine (FIG. 3c). Among the lysine
analogs, only 1, 2, and 4 significantly repress β-galactosidase
expression, confirming that they can repress natural lysC and, most
likely, yvsH expression.

[0323]The minimal inhibitory concentration (MIC) of 1, 2, and 4 that
prevent growth of B. subtilis are similar to the MIC measured for AEC on
this and other bacteria (reference Japanese paper). Notably, the relative
concentrations of lysine, 1, and 2 required to completely repress
expression (FIG. 3d) correspond well with their relative KD and MIC
values. Furthermore, at their respective MICs, 1 and 2 completely repress
reporter gene expression. The strong correlation among KD, reporter
gene repression, and antibacterial activity is consistent with a
mechanism wherein riboswitch-mediated gene repression is responsible for
the inhibitory activity of these compounds.

[0324]To more fully characterize the mechanism of inhibition, B. subtilis
strains were cultivated that are resistant to 2. Resistance to 2 was
examined because this compound is a commercially available representative
of the compounds that exhibit strong binding to the riboswitch and a good
MIC value. Using serial passage (see Methods), 24 bacterial colonies were
isolated that exhibit at least 9-fold higher MIC values for 2, and DNAs
corresponding to the lysC and yvsH riboswitches from these resistant
bacteria were amplified and sequenced. Not surprisingly, no mutations
were observed in the yvsH riboswitches of these bacteria. Since these
resistant bacteria were evolved in a defined minimal media without lysine
supplementation, there was no selective pressure to derepress the
expression of the lysine transporter coded by yvsH. Remarkably, every
resistant colony had a single mutation in the lysC riboswitch (FIG. 4a).
Among the resistant colonies, 21 had a G to A mutation in P4 (M1), and
three had an A deletion in the loop E motif of P2 (M2). Importantly, both
mutations also confer resistance to 1 and 4 (FIG. 4b), implying that the
compounds might have a common mechanism of action. When cloned upstream
of the β-galactosidase reporter, constructs containing either the M1
or M2 mutation derepress gene expression, even at lysine concentrations
as high as 5 mM. Moreover, because fully active β-galactosidase is
still expressed when cells are grown in the presence of high
concentrations of 1 and 2, it is unlikely that incorporation of these
compounds into proteins is the cause for growth inhibition.

[0325]To understand how the mutations disable the lysine riboswitch,
in-line probing assays were performed with the lysC receptor region
carrying the M1 or M2 mutations. In the absence of ligand, the M1
mutation destabilizes P4 at room temperature and this effect is more
pronounced at 37° C. (FIG. 4c). Likewise, in the absence of
ligand, the M2 mutation destabilizes the loop E structure. Surprisingly,
these structural defects only modestly decrease the KD for lysine
(FIG. 4b). It is possible that, like other riboswitches, (Wickisier
20051; Wickisier 20052) the speed at which the ligand binds the
lysine riboswitch, rather than its binding affinity, determines whether
gene repression occurs. If that is the case, the mutations disrupt gene
regulation by slowing ligand association.

[0326]To directly investigate how the M1 and M2 deformations alter gene
regulation, in vitro transcription assays were conducted with each
variant. In the absence of a ligand, both mutations decrease the
termination efficiency compared to the wild-type riboswitch (FIG. 4d).
Most likely, each mutation causes a fraction of the receptors to fold
into an inactive form that can not form the terminator structure. This
effect is not rescued by adding a saturating concentration of lysine
(FIG. 4d,e) supporting the assertion that a fraction of the mutated
riboswitches is inactive. In addition to causing a folding defect, the
mutations result in an increased concentration of ligand needed to induce
termination (T50, FIG. 4e). This shows that each mutation also
affects ligand binding by the receptor, even within the properly folded
population. Because the in vitro binding affinities are not dramatically
shifted, this effect most likely reflects a decreased rate of ligand
association. In summary, the results indicate that mutations that impair
the gene regulatory function of a lysine riboswitch confer resistance to
antibacterial lysine analogs.

[0327]Collectively, these data demonstrate that the antibacterial lysine
mimetics 1, 2, and 4 function, at least in part, by lysine
riboswitch-mediated repression of aspartokinase II in B. subtilis.

[0328]These compounds can also inhibit bacterial growth in an infection
setting. Because many bacteria have an isozyme for aspartokinase II that
is not regulated by the riboswitch (FIG. 6), (Zhang 1990) full repression
of lysC might not sufficiently quell lysine biosynthesis to inhibit
growth within a host environment. However, in a few species, such as
Bacillus cereus and Bacillus anthracis, an additional copy of the
riboswitch regulates the diaminopimelate decarboxylase gene (lysA in B.
subtilis, FIG. 1b), for which no alternate pathway exists (Rodionov
2003). Accordingly, 1 inhibits the growth of B. cereus 13-fold more
severely than that of B. subtilis in minimal medium, implying that
repression of genes that are more critical to survival is more
detrimental to growth. Regardless, neither B. subtilis nor B. anthracis
is inhibited by 512 μg ml-1 of 1, or 2 in rich media (see Methods
for details). Most likely, dipeptide (Higgins 1986) or other
non-regulated amino acid transporters can supply enough lysine from the
media, even when lysine biosynthesis is completely repressed. This result
shows that compounds that exclusively target the lysine riboswitch are
not necessarily potent against pathogens that can glean lysine from the
host.

[0329]In conclusion, evidence is provided that the lysine riboswitch can
serve as an antibacterial drug target in minimal media. It was also found
that AEC, originally characterized in 1958 (Shiota 1958) inhibits
bacterial growth by targeting the lysine riboswitch (Sudarsan 2006).
Combined with the recent discovery that the antibacterial activity of
pyrithiamin, also established decades ago (Woolley 1943) targets the
thiamine pyrophosphate-binding riboswitch (Sudarsan 2005) this work
underscores the generality of targeting riboswitches with antibacterial
drugs.

Methods

[0330]Chemicals, oligonucleotides, and bacterial strains. L-lysine,
L-4-oxalysine, L-homoarginine, L-N2-acetyllysine,
L-N6-acetyllysine, L-N6-1-iminoethyllysine, L-3-aminotyrosine,
L-2-amino-3-(2-aminobenzoyl)-propionic acid, and
L-N6-trimethyllysine were purchased from Sigma.
DL-trans-2,6-diamino-4-hexenoic acid, DL-5-oxolysine, and
L-N2-methyllysine were purchased from Bachem. Decoyinine was
purchased from MP Biomedicals. Oligonucleotides were synthesized by the
HHMI Keck Foundation Biotechnology Resource Center at Yale University.
All B. subtilis strains were obtained from the Bacillus Genetic Stock
Center (The Ohio State University), with the exception of the M1 and M2
strains that were generated in this study.

[0331]L-3-[(2-aminoethyl)-sulfonyl]-alanine was prepared similarly to a
previously described method (Toennies 1941). Perchloric acid (70%, 4.1
ml, 47.5 mmol) was added to a solution of ammonium molybdate (586 mg, 3.0
mmol) in 15 ml of water, and the solution was heated at 100-110°
C. until a white solid formed. After cooling to 25° C., the
mixture was filtered and the filtrate was treated with
L-2-aminoethylcysteine (300 mg, 1.5 mmol) followed by hydrogen peroxide
(30%, 11.7 ml, 0.122 mol). The mixture was stirred at 25° C.
overnight and loaded into Dowex 50WX resin (H.sup.+ form). After washing
the resin with H2O, the product was collected by eluting with 2N
NH4OH and then concentrated in vacuo to give the product (304.4 mg,
87.5%): 1H NMR (400 MHz, D2O) δ 2.81 (m, 2H), 3.04 (m,
2H), 3.16 (t, 2H), 3.88 (t, 1H).

[0332]In-line probing assays. The 179 lysC RNA construct used for in-line
probing assays was prepared by in vitro transcription using a template
generated from whole-cell PCR of the appropriate B. subtilis strains (1A1
or resistant strains). RNA transcripts were dephosphorylated,
5'-32P-labeled, and subsequently subjected to in-line probing using
protocols similar to those described previously (Soukup 1999). For each
reaction, approximately 1 nM of labeled RNA was incubated for 39-48 h at
room temperature or 16-20 h at 37° C. in a 10 μl solution
containing 50 mM Tris (pH 8.3 at 25° C.), 20 mM MgCl2, and
100 mM KCl in the absence or presence of 1 nM to 6 mM of lysine or each
analog as indicated for each experiment. Denaturing 10% polyacrylamide
gel electrophoresis (PAGE) was used to separate spontaneous cleavage
products, which were detected and quantitated using a GE Healthcare
PhosphorImager and ImageQuant NT software.

[0333]The KD for each ligand was derived by quantifying the amount of
RNA cleaved at each nucleotide position over a range of ligand
concentrations. For each region where modification was observed (A, B,
and C in FIG. 2b), the fraction of RNA cleaved at each ligand
concentration was calculated by assuming that the maximal extent of
cleavage is observed in the absence of ligand and the minimal cleavage is
observed in the presence of the highest ligand concentration. The
apparent KD was determined by fitting the plot of the fraction
cleaved, x, versus the ligand concentration, [L], to the following
equation: χ=KD/([L]+KD), using SigmaPlot 9 software.

[0335]Sporulation assays. Sporulation effects were determined as described
elsewhere (Lazazzera 1997). Briefly, an overnight culture of B. subtilis
168 strain 1A1 in GMM was diluted by a factor of 30 into GMM supplemented
with 20 mM glutamate and grown at 37° C. with shaking to an
A600 of 0.5-0.7. After adding an aliquot of the indicated lysine
analog, sporulation was induced by adding decoyinine to a final
concentration of 500 μg/ml. After growing for an additional 22-24 h,
the efficiency at which cultures formed viable spores was measured by
plating an aliquot of the culture onto TBAB either before or after
heating at 80° C. for 20 min and quantifying the surviving
colonies.

[0336]In vivo reporter gene expression assays. The lysC 5'-UTR from a
wild-type B. subtilis strain or a representative of the resistant M1 or
M2 strains was PCR amplified, ligated upstream of a β-galactosidase
reporter gene, and integrated into the genome of B. subtilis following
methods described previously (Sudarsan 2006). Briefly, nucleotides--399
to ˜17 relative to the lysC translation start site were amplified
as an EcoRI-BamHI fragment by whole-cell PCR of each strain. The PCR
products were cloned into pDG1661 (Guerot-Fleury 1996) immediately
upstream of the lacZ reporter gene, with integrity confirmed by
sequencing. The resulting pDG1661 variants were transformed into the amyE
locus of B. subtilis strain 1A40 using standard protocols (Jarmer 2002)
and correct transformants were selected by screening for chloramphenicol
(5 μg/ml) resistance and spectinomycin (50 μg/ml) sensitivity.

[0337]β-galactosidase expression levels of each strain were measured
as described previously (Sudarsan 2006). Briefly, cells were grown
overnight with shaking at 37° C. in GMM supplemented with 50
μg/ml each of tryptophan, methionine and lysine. The following day,
the cells were centrifuged, and the pellet was resuspended in GMM
supplemented with 50 μg/ml each of tryptophan and methionine. The
resuspended cells were diluted by a factor of 10 into the same medium
supplemented with lysine or a lysine analog at the indicated
concentration. After growing for 3 h at 37° C.,
β-galactosidase assays were performed using a standard protocol.

[0338]β-galactosidase expression as a function of ligand
concentration was determined similarly, except that cell growth and
quantitation of expression were performed in 96-well microplates.
Briefly, a culture of each strain at a starting A595 of 0.6 was
grown for 3 h at 37° C. in 150 μl of GMM supplemented with 50
μg/ml tryptophan and methionine and varying ligand concentrations.
After recording the absorbance at 595 nm using a Beckman Coulter DTX 880
plate reader, the cells were permeabilized by mixing 100 of each culture
into 0.5 ml Z-buffer (100 mM Na2HPO4 [pH 7.0 at 25° C.],
10 mM KCl, 1 mM MgSO4, and 50 mM β-mercaptoethanol), 10 μl
0.1% SDS, and 40 μl of chloroform per well of a polypropylene
microplate. After allowing the solution to settle, 150 μl was
transferred to a separate plate and incubated for 20 min at 25° C.
with 25 μl ortho-nitrophenyl-β-galactoside, followed by
quantitation of the absorbance at 414 nm and 550 nm and calculation of
Miller units as previously reported for riboswitch gene repression assays
(Sudarsan 2006).

[0339]Evolution of L-4-oxalysine-resistant strains. Resistant mutants were
cultivated using serial passage (Kawasaki 1969). A fresh overnight
culture of B. subtilis 168 strain 1A1 in GMM supplemented with 50
μg/ml tryptophan was diluted by a factor of 100 into the same medium
with 100 μM L-4-oxalysine (2) and grown at 37° C. with shaking,
until the culture reached saturation. A 10 μl aliquot was then diluted
into 1 ml GMM containing 100 μM L-4-oxalysine, and this process was
repeated until, after six passages, the culture reached saturation at the
same rate as a culture with no added compound. After plating onto
tryptone blood agar base (TBAB), the genomic regions encompassing the
5'-UTRs of the lysC gene (-486 to +110 relative to the translation start
site) and the yvsH gene (-376 to +1564) were amplified and sequenced from
24 of the resistant isolates.

[0340]In vitro transcription termination assays. Single-round
transcription termination assays were conducted following protocols
adapted from a previously described method (Landick 1996). The DNA
templates covered the region -390 to -17 (relative to the start of
translation) of the B. subtilis lysC gene, with a point mutation (C6G of
the RNA) to eliminate C residues on the nascent RNA before nucleotide
position 17 and were generated by whole-cell PCR of B. subtilis 168
strain 1A1 or the corresponding resistant strains. To initiate
transcription and form C17-halted complexes, each sample was incubated at
37° C. for 10 min and contained 1 pmole DNA template, 0.2 mM ApA
dinucleotide, 1 μM each of ATP, GTP, and UTP, plus 2 μCi
5'-[α-32P]-UTP, and 0.4 U E. coli RNA polymerase holoenzyme
(Epicenter) in 10 μl of 80 mM Tris-HCl (pH 8.0 at 26° C.), 20
mM NaCl, 14 mM MgCl2, 0.1 mM EDTA and 0.01 mg/ml BSA. Halted
complexes were restarted by the simultaneous addition of 10 μM each of
the four NTPs, 0.2 mg/ml heparin to prevent re-initiation, and different
concentrations of ligand as indicated to yield a final volume of 12.5
μl in a buffer containing 150 mM Tris-HCl (pH 8.0 at 26° C.),
20 mM NaCl, 14 mM MgCl2, 0.1 mM EDTA and 0.01 mg/ml BSA. Reactions
were incubated for an additional 20 min at 37° C., and the
products were separated by denaturing 10% PAGE followed by quantitation
as described above.

TABLE-US-00002
TABLE 2
Sporulation efficiency of B. subtilis when grown in the presence
of lysine analogs, determined as described elsewhere. (Lazazzera
1997). The data are expressed as the number of viable colony
forming spores formed after heat shock relative to the number of
viable colony forming units present before heat shock. Four analogs
that affected cell growth were tested for sporulation effects to provide
two examples of compounds that are bound by the riboswitch and two
examples of compounds that are not bound by the riboswitch.
% Viable
spore formation
Analog Concentration (mM) WT M2
No ligand -- 71 19
1 0.2 7 ND1
2 0 17
2 0.2 71 ND
2 6 65
8 2 47 9
9 2 75 75
1ND designates not determined.

[0341]It is understood that the disclosed method and compositions are not
limited to the particular methodology, protocols, and reagents described
as these may vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention which will be
limited only by the appended claims.

[0342]It must be noted that as used herein and in the appended claims, the
singular forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to "a
riboswitch" includes a plurality of such riboswitches, reference to "the
riboswitch" is a reference to one or more riboswitches and equivalents
thereof known to those skilled in the art, and so forth.

[0343]"Optional" or "optionally" means that the subsequently described
event, circumstance, or material may or may not occur or be present, and
that the description includes instances where the event, circumstance, or
material occurs or is present and instances where it does not occur or is
not present.

[0344]Ranges may be expressed herein as from "about" one particular value,
and/or to "about" another particular value. When such a range is
expressed, also specifically contemplated and considered disclosed is the
range from the one particular value and/or to the other particular value
unless the context specifically indicates otherwise. Similarly, when
values are expressed as approximations, by use of the antecedent "about,"
it will be understood that the particular value forms another,
specifically contemplated embodiment that should be considered disclosed
unless the context specifically indicates otherwise. It will be further
understood that the endpoints of each of the ranges are significant both
in relation to the other endpoint, and independently of the other
endpoint unless the context specifically indicates otherwise. Finally, it
should be understood that all of the individual values and sub-ranges of
values contained within an explicitly disclosed range are also
specifically contemplated and should be considered disclosed unless the
context specifically indicates otherwise. The foregoing applies
regardless of whether in particular cases some or all of these
embodiments are explicitly disclosed.

[0345]Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as commonly understood by one of skill in
the art to which the disclosed method and compositions belong. Although
any methods and materials similar or equivalent to those described herein
can be used in the practice or testing of the present method and
compositions, the particularly useful methods, devices, and materials are
as described. Publications cited herein and the material for which they
are cited are hereby specifically incorporated by reference. Nothing
herein is to be construed as an admission that the present invention is
not entitled to antedate such disclosure by virtue of prior invention. No
admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and applicants
reserve the right to challenge the accuracy and pertinency of the cited
documents. It will be clearly understood that, although a number of
publications are referred to herein, such reference does not constitute
an admission that any of these documents forms part of the common general
knowledge in the art.

[0346]Throughout the description and claims of this specification, the
word "comprise" and variations of the word, such as "comprising" and
"comprises," means "including but not limited to," and is not intended to
exclude, for example, other additives, components, integers or steps.

[0347]Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the method and compositions described herein.
Such equivalents are intended to be encompassed by the following claims.